Method for detecting lysosomal storage diseases

ABSTRACT

A method for detecting lysosomal storage diseases including the steps of performing an assay for a single species of glycosaminoglycan contained in a specimen and correlating results of the assay with lysosomal storage diseases. A body fluid such as urine or blood can be employed as a specimen. The assay can be performed by use of a polypeptide that is capable of specifically binding to a glycosaminoglycan-containing molecule. The polypeptide may be an antibody, or a polypeptide having an antigen-binding site of an antibody.

TECHNICAL FIELD

The present invention relates to a method for detecting lysosomalstorage diseases and to a kit therefor.

BACKGROUND ART

The following abbreviations are used throughout the presentspecification:

-   -   GAG: glycosaminoglycan    -   KS: keratan sulfate    -   HS: heparan sulfate    -   CS: chondroitin sulfate    -   CS-4S: chondroitin-4-sulfate    -   CS-6S: chondroitin-6-sulfate    -   DS: dermatan sulfate (also called chondroitin sulfate B)    -   GSD I: glycogen storage disease type 1    -   GSD II: glycogen storage disease type 2 (Pompe disease)    -   Hep: heparin    -   HA: hyaluronic acid    -   LIPO: lipofuscinoses    -   MPS: mucopolysaochaxidoses    -   ML: mucolipidoses    -   MLD: metachromatic leukodystropy    -   NP: Niemann-Pick diseases    -   TS: Tay-Sachs disease

Lysosomal storage diseases are diseases caused by abnormality of enzymespresent in lysosomes.

Mucopolysaccharidoses are kinds of lysosomal storage diseases and form aclass of hereditary diseases caused by deficiency of enzymes (loweredactivity of enzymes) involved in degradation metabolism of GAGs. Inaccordance with the species of the defective enzyme, GAG of a specificspecies is known to be accumulated in tissues and excreted into bodyfluids. Clinical manifestations of mucopolysaccharidoses arediversified, but most cases involve coarse facial expression, dysostosismultiplex, and visceromegaly. In same oases, hypacusia, cardiovasculardisorders, and mental retardation are also observed.

Table 1-1 shows the known relations between types ofmucopolysaccharidoses and corresponding GAGs that are accumulated (see,for example, “The Metabolic and Molecular Bases of Inherited Disease”,7th edn., Scriver C R, Beaudet A L, Sly W S, Valle D (eds.), 1995,McGraw-Hill, New York).

TABLE 1-1 mucopolysaccharidoses Substance Type Disease Name EnzymeDeficiency Stored I H Hurler α-L-iduronidase HS, DS Syndrome I S Scheiesame as above HS, DS Syndrome I H/S Hurler-Scheie same as above HS, DSComplex II A Hunter iduronate sulfatase HS, DS Syndrome (Severe) II BHunter same as above HS, DS Syndrome (Mild) III A Sanfilippo heparanN-sulfatase HS Syndrome A III B Sanfilippo α-N-acetylglucosaminidase HSSyndrome B III C Sanfilippo acetyl CoA: α-glucosaminide N- HS Syndrome Cacetyltransferase III D Sanfilippo N-acetylglucosamine-6-sulfatase HSSyndrome D IV A Morquio galactosamine-6-sulfatase KS Syndrome A IV BMorquio β-galactosidase KS Syndrome B VI A Maroteaux- arylsulfatase B DSLamy Syndrome (Severe) VI B Maroteaux- same as above DS Lamy Syndrome(Mild) VII β- β-glucuronidase HS, DS, glucuronidase CS-4S, deficiencyCS-6S

TABLE 1-2 mucolipidoses Type Disease Name Enzyme Deficiency SubstanceStored II I-Cell disease N-acetylglucosamine-1- Inclusion bodyphosphotransferase III Pseudo-Hurier same as above (Mild) Inclusion bodyPolydystrophy

However, until the present invention, it has remained unknown that eachcase of the mentioned mucopolysaccharidoses not only involves excretionin body fluids of the GAG species shown in Table 1-1, but also involvesexcretion in body fluids of large amounts of other species of GAGs.

Japanese Patent Application Laid-Open (kokai) No. 10-153600 discloses anassay method in which a solid phase to which a first receptor (anti-GAGantibodies such as anti-KS antibody, anti-CS antibody, anti-HS antibodyor the like) is immobilized is brought into contact with a specimencontaining a first ligand (GAG such as KS, CS, HS or the like), andformation of a complex between the first receptor and the first ligandis detected by a first-labeling-substance-labeled first receptor, tothereby assay the first ligand contained in the specimen. Thispublication also discloses that the method facilitates primary screeningof GAG-related diseases (including mucopolysaccharidoses such asMorquio's syndrome and Hurler's syndrome).

However, Japanese Patent Application Laid-Open (kokai) No. 10-153600neither discloses nor suggests that each case of the aforementionedmucopolysaccharidoses involves, in addition to the GAG species shown inTable 1-1 being excreted, excretion into body fluid of large amounts ofother species of GAGs. Moreover, this publication neither discloses norsuggests whether measurement of GAG of a single species enablesdetection of all types of mucopolysaccharidoses, regardless of theclassification (type) of mucopolysaccharidoses.

Mucolipidoses are also kinds of lysosomal storage diseases and arediseases that show similar clinical symptoms to those ofmucopolysaccharidoses. It is known that types of the mucolipidoses areas shown in Table 1-2.

Also, GM1 gangliosidoses and fucosidoses are also kinds of lysosomalstorage diseases. GM1 gangliosidoses are diseases in which GM1ganglioside and β-galactose residue-containing oligosaccharides andglycoproteins are accumulated due to impediment in β-galactosidase, andfucosidoses are diseases in which oligosaccharides and glycoproteinshaving α-fucose residues are accumulated due to impediment inα-fucosidase.

In addition, galactosialidoses are also kinds of lysosomal storagediseases and are diseases in which sialyloligosaccharides and substancessimilar to the case of GM1 gangliosidoses are accumulated due toimpediment in β-galactosidase and α-neuraminidase, and impediment incathepsin A which is involved in the stabilization of these enzymes.

Furthermore, the following diseases are also kinds of lysosomal storagediseases.

Metachromatic leukodystrophy is a disease in which sulphatides areaccumulated due to impediment in arylsulfatase A.

Niemann-Pick diseases are diseases in which sphingomyelin isaccumulated. Niemann-Pick type B is due to impediment in acidsphingomyelinase, and Niemann-Pick type C is due to cholesterolesterification defect.

Tay-Sachs disease is disease in which GM2 ganglioside is accumulated dueto impediment in α-subunit of N-acetyl-β-D-glucosaminidase A.

Sandhoff disease is disease in which GM2 ganglioside is accumulated dueto impediment in β-subunit of N-acetyl-β-D-glucosaminidase A and B.

GM2 gangliosidoses are diseases in which GM2 ganglioside is accumulateddue to impediment in GM2 activator protein.

Krabbe disease is disease in which galactocerebroside is accumulated dueto impediment in β-D-galactocerebrosidase.

Fabry disease is disease in which globosides are accumulated due toimpediment in α-D-galactosidase.

Gaucher diseases are diseases in which glucosylceramide is accumulateddue to impediment in β-D-glucocerebrosidase.

Glycogen storage disease type 1 is disease in which glycogen isaccumulated due to impediment in glucose-6-phosphatase.

Glycogen storage disease type 2 (also called Pompe disease) is diseasein which glycogen is accumulated due to impediment in α-D-glucosidase.

Lipofuscinoses are caused by impediment in palmitoyl-proteinthioesterase or tripeptidyl amino peptidase-I.

It has not been known that GAG is also discharged in a large amount intobody fluids in these diseases.

Hereinafter, mucopolysaccharidoses, mucolipidoses, GM1 gangliosidoses,fucosidoses, galactosialidoses, metachromatic_leukodystropy,Niemann-Pick diseases, Tay-Sachs disease, Sandhoff disease, GM2gangliosidoses, Krabbe disease, Fabry disease, Gaucher diseases,glycogen storage diseases and lipofuscinoses are referred to as“mucopolysaccharidoses, etc.”

In general, mucopolysaccharidoses, etc. are asymptomatic in newborns,but onset thereof becomes clear by manifestations including arrestedheight gain, abnormal development of bones, and growth of shaggy hairduring infancy or childhood. In some cases, although subjects are normalduring neonatal periods, mental retardation gradually progresses overyears. Therefore, diagnosis of mucopolysaccharidoses, etc. in an earlynewborn stage during which no clinical syndromes are manifested maypossibly prevent mental retardation, etc., through early enzymereplacement therapy, genetic treatment, or bone marrow transplantation.Therefore, diagnosis of mucopolysaccharidoses, etc. is desirablyperformed for all newborns.

However, in Japan, for example, the number of newborns per year exceeds1,000,000, and the frequency of onset of mucopolysaccharidoses, etc. isas low as one per 40,000 to 50,000, and since current diagnosistherefor, which detects deficiency or abnormality of enzymes, iscumbersome and expensive, demand exists for an accurate screening methodto be performed before such an expensive diagnosis. In other words, ifthere can be provided a method for detecting patients sufferingmucopolysaccharidoses, etc. with high accuracy, with high sensitivity,conveniently, quickly, and at low cost, without overlooking any patientsof mucopolysaccharidoses, etc., presence or absence ofmucopolysaccharidoses, etc. can be detected in all newborns, and thusprecise, definite diagnosis of every patient of mucopolysaccharidoses,etc. can be attained at an early stage of the disease.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide a veryaccurate, sensitive method for detecting lysosomal storage diseaseswhich can be performed conveniently, quickly, and at low cost. Anotherobject of the invention is to provide a kit for detecting lysosomalstorage diseases.

The present inventors have carried out extensive research in an attemptto attain the above objects, and have found the following: in spite ofconventional understanding that, in accordance with the type(classification) of mucopolysaccharidosis, only GAG of a specificspecies is excreted into body fluids in large amounts, in reality otherspecies of GAGs are also excreted abundantly. Moreover, the presentinventors found that GAG is also excreted into body fluids in largeamounts in mucopolysaccharidoses, mucolipidoses, GM1 gangliosidoses,fucosidoses, galactosialidoses, metachromatic leukodystropy,Niemann-Pick diseases, Tay-Sachs disease, Sandhoff disease, GM2gangliosidoses, Krabbe disease, Fabry disease, Gaucher diseases,glycogen storage diseases and lipofuscinoses. On the basis of thisfinding, the inventors have achieved a highly accurate, highlysensitive, convenient, effective, inexpensive method and kit fordetecting lysosomal storage diseases, thus leading to completion of theinvention.

Accordingly, the present invention provides a method for detectinglysosomal storage diseases, comprising the steps of performing an assayfor GAG of a single species contained in a specimen, and correlatingresults of the assay with lysosomal storage diseases (hereinafter themethod is called “the method of the invention” or “the present method”).

Preferably, the “lysosomal storage diseases” are at least one diseaseselected from mucopolysaccharidoses, mucolipidoses, GM1 gangliosidoses,fucosidoses, galactosialidoses, metachromatic leukodystropy,Niemann-Pick diseases, Tay-Sachs disease, Sandhoff disease, GM2gangliosidoses, Krabbe disease, Fabry disease, Gaucher diseases,glycogen storage diseases and lipofuscinoses.

Preferably, the specimen is a body fluid, with urine or blood being morepreferred.

Preferably, the assay of the mentioned single species of GAG isperformed by use of a polypeptide that is capable of specificallybinding to a GAG-containing molecule.

Preferably, the assay comprises the following steps (1) and (2):

(1) a step for forming a sandwich-like complex by bringing “a solidphase to which a first polypeptide capable of specifically binding to aGAG-containing molecule is immobilized”, “a specimen”, and “a secondpolypeptide capable of specifically binding to a GAG-containingmolecule” into contact with one another, the sandwich-like complex beingconstituted by “said first polypeptide immobilized onto the solidphase—GAG-containing molecule in the specimen—second polypeptide”; and

(2) a step for detecting the sandwich-like complex formed in step (1).

More preferably, the assay comprises the following steps (1), (2), and(3):

(1) a step for forming a complex by bringing “a solid phase to which afirst polypeptide capable of specifically binding to a GAG-containingmolecule is immobilized” into contact with “a specimen”, the complexbeing constituted by “first polypeptide immobilized onto the solidphase—GAG-containing molecule in the specimen”;

(2) a step for forming a sandwich-like complex by bringing theabove-described solid phase into contact with “a second polypeptidecapable of specifically binding to a GAG-containing molecule”, thesandwich-like complex being constituted by “said first polypeptideimmobilized onto the solid phase—GAG-containing molecule in thespecimen—second polypeptide”; and

(3) a step for detecting the sandwich-like complex formed in step (2).

Preferably, the “second polypeptide” is labeled with a labelingsubstance or, alternatively, is capable of being labeled with a labelingsubstance.

Preferably, the assay comprises the following steps (1) and (2):

(1) a step for forming first and second complexes by bringing “a thirdpolypeptide capable of specifically binding to a GAG-containingmolecule”, “a specimen”, and “a solid phase to which a GAG-containingmolecule is immobilized” into contact with one another, the firstcomplex being constituted by “GAG-containing molecule immobilized onto asolid phase—third polypeptide” and the second complex being constitutedby “GAG-containing molecule is the specimen—third polypeptide”; and

(2) a step for detecting at least one of the complexes formed in step(1), the first complex being “GAG-containing molecule immobilized onto asolid phase—third polypeptide” and the second complex being“GAG-containing molecule in the specimen—third polypeptide.”

More preferably, the assay comprises the following steps (1) to (3):

(1) a step for forming a first complex by bringing into contact “a thirdpolypeptide capable of specifically binding to a GAG-containingmolecule” and “a specimen”, the first complex being constituted by“third polypeptide—GAG-containing molecule in the specimen”;

(2) a step for forming a second complex by bringing “the solid phase towhich a GAG-containing molecule is immobilised” into contact with amixture resulting from step (1) which contains “the first complex” and“a third polypeptide that participated in formation of the firstcomplex”, the second complex being constituted by “GAG-containingmolecule immobilized onto the solid phase—third polypeptide”; and

(3) a step for detecting the second complex formed in step (2).

Detection of the second complex is preferably carried out by use of afourth polypeptide capable of being specifically binding to the thirdpolypeptide and having been labeled with, or being capable of beinglabeled with, a labeling substance.

Any of the polypeptides employed in the above methods is preferably anantibody or a polypeptide having an antigen-binding site of an antibody.

In the method of the present invention, preferably, the “GAG of a singlespecies” is a GAG having a sulfate group. The GAG having a sulfate groupis preferably KS, HS, CS, or DS. In the present method, preferred arethe cases where the “GAG having a sulfate group” is KS, andsimultaneously, the “mucopolysaccharidoses” are of one or more typesselected from among mucopolysaccharidosis types I, II, III, VI, and VII.Also preferred are the cases where the “GAG having a sulfate group” isKS, and the mucolipidoses are of one or more types selected from amongmucolipidosis types II and Also preferred are the cases where the “GAGhaving a sulfate group” is HS, and simultaneously, the“mucopolysaccharidoses” are of one or more types selected from amongmucopolysaccharidosis types IV and VI; the cases where the “GAG having asulfate group” is HS, and the lysosomal storage diseases are of one ormore diseases selected from among mucolipidoses, metachromaticleukodystropy, Niemann-Pick diseases, Tay-Sachs disease, Sandhoffdisease, GM2 gangliosidoses, Krabbe disease, Fabry disease, Gaucherdiseases, glycogen storage diseases and lipofuscinoses; the cases wherethe “GAG having a sulfate group” is CS, and simultaneously, the“mucopolysaccharidoses” are of one or more types selected from amongmucopolysaccharidosis types I, II, III, IV, and VI; and the cases wherethe “GAG having a sulfate group” is DS, and simultaneously, the“mucopolysaccharidoses” are of one or more types selected from amongmucopolysaccharidosis types III and IV.

The present invention also provides a kit for detecting at least onedisease selected from mucopolysaccharidoses, mucolipidoses, GM1gangliosidoses, fucosidoses, galactosialidoses, metachromaticleukodystropy, Niemann-Pick diseases, Tay-Sachs disease, Sandhoffdisease, GM2 gangliosidoses, Krabbe disease, Fabry disease, Gaucherdiseases, glycogen storage diseases and lipofuscinoses comprising thefollowing components and used for detecting at least one diseaseselected from mucopolysaccharidoses, mucolipidoses, GM1 gangliosidoses,fucosidoses, galactosialidoses, metachromatic leukodystropy,Niemann-Pick diseases, Tay-Sachs disease, Sandhoff disease, GM2gangliosidoses, Krabbe disease, Fabry disease, Gaucher diseases,glycogen storage diseases and lipofuscinoses on the basis of assayresults of GAG of a single species contained in a specimen (hereinafterthe kit is called “the kit of the invention” or “the present kit”):

(A) a solid. phase to which a first polypeptide capable of specificallybinding to a GAG-containing molecule is immobilized; and

(B) a second polypeptide capable of specifically binding to aGAG-containing molecule and having been labeled with, or being capableof being labeled with, a labeling substance.

Alternatively, the kit of the present invention may comprise thefollowing components:

(A) a solid phase to which a GAG-containing molecule is immobilized;

(B) a third polypeptide capable of specifically binding to aGAG-containing molecule; and

(C) a fourth polypeptide capable of specifically binding to the thirdpolypeptide, and having been labeled with, or being capable of beinglabeled with, a labeling substance.

Preferably, any of these polypeptides is an antibody or a polypeptidehaving an antigen-binding site of an antibody.

In the kit of the present invention, preferably, the “GAG of a singlespecies” is a GAG having a sulfate group. The GAG having a sulfate groupis preferably KS, HS, CS, or DS. In the present kit, preferred are thecases where the “GAG having a sulfate group” is KS, and simultaneously,the “mucopolysaccharidoses” are of one or more types selected from amongmucopolysaccharidosis types I, II, III, VI, and VII. Also preferred arethe cases where the “GAG having a sulfate group” is KS, and themucolipidoses are of one or more types selected from among mucolipidosistypes II and III. Also preferred are the cases where the “GAG having asulfate group” is HS, and simultaneously, the “mucopolysaccharidoses”are of one or more types selected from among mucopolysaccharidosis typesIV and VI; the cases where the “GAG having a sulfate group” is HS, andthe diseases are of one or more diseases selected from amongmucolipidoses, metachromatic leukodystropy, Niemann-Pick diseases,Tay-Sachs disease, Sandhoff disease, GM2 gangliosidoses, Krabbe disease,Fabry disease, Gaucher diseases, glycogen storage diseases andlipofuscinoses; the cases where the “GAG having a sulfate group” is CS,and simultaneously, the “mucopolysaccharidoses” are of one or more typesselected from among mucopolysaccharidosis types I, II, III, IV, and VI;and the cases where the “GAG having a sulfate group” is DS, andsimultaneously, the “mucopolysaccharidoses” are of one or more typesselected from among mucopolysaccharidosis types III and IV.

Various modes of the present invention will next be described.

<1> Method of the Present Invention

The present invention contemplates a detection method for lysosomalstorage diseases, and comprises the steps of measuring GAG of a singlespecies contained in a specimen and correlating the measurement resultswith lysosomal storage diseases.

Preferably, the present invention contemplates a detection method for atleast one disease selected from mucopolysaccharidoses, mucolipidoses,GM1 gangliosidoses, fucosidoses, galactosialidoses, metachromaticleukodystropy, Niemann-Pick diseases, Tay-Sachs disease, Sandhoffdisease, GM2 gangliosidoses, Krabbe disease, Fabry disease, Gaucherdiseases, glycogen storage diseases and lipofuscinoses and comprises thesteps of measuring GAG of a single species contained in a specimen andcorrelating the measurement results with at least one disease selectedfrom mucopolysaccharidoses, mucolipidoses, GM1 gangliosidoses,fucosidoses, galactosialidoses, metachromatic leukodystropy,Niemann-Pick diseases, Tay-Sachs disease, Sandhoff disease, GM2gangliosidoses, Krabbe disease, Fabry disease, Gaucher diseases,glycogen storage diseases and lipofuscinoses.

A characteristic feature of the method of the present invention residesin the detection of mucopolysaccharidoses through measurement of GAG of“a single species”, rather than through measurement of GAG of “aplurality of species” contained in a specimen. It has hitherto beenaccepted that depending on the classification (type) ofmucopolysaccharidoses, different species of GAGs are accumulated andexcreted. Therefore, quite naturally, according to conventionalunderstanding, even when the measurement of a certain species of GAG(e.g., KS) is negative, presence of mucopolysaccharidoses cannot bedenied until measurements of other species of GAG (e.g., HS, CS, DS orthe like) support the negative results. That is, conventionally, inorder to detect mucopolysaccharidoses, a plurality of species of GAGsmust be measured for a single specimen.

In contrast, the method of the present invention enables correlationwith mucopolysaccharidoses to be established by the measurement of onlyGAG of a single species. Furthermore, the method of the presentinvention enables correlation with not only mucopolysaccharidoses butalso at least one disease selected from mucolipidoses, GM1gangliosidoses, fucosidoses, galactosialidoses, metachromaticleukodystropy, Niemann-Pick diseases, Tay-Sachs disease, Sandhoffdisease, GM2 gangliosidoses, Krabbe disease, Fabry disease, Gaucherdiseases, glycogen storage diseases and lipofuscinoses.

Although no particular limitation is imposed on the specimen that can beused in the present invention, body fluid is preferred. No particularlimitation is imposed on the body fluid, so long as it contains, orpossibly contains, GAG accumulated as a result of lysosomal storagediseases. Examples of the body fluid include urine, blood (as usedherein, the term “blood” is used to encompasses serum and plasma),saliva, sweat, tears, synovial fluid, cartilage extracts, andsupernatants of cell cultures. Of these, urine and blood are preferred,as they are easily collected from newborns and in fact are routinelycollected for usual newborn health check items.

When blood is used as the body fluid, the collected blood sample may beused as is without any treatment, or serum or plasma derived from thecollected blood sample may be used. Preferably, serum or plasma isemployed. Alternatively, hydrophilic components may be extracted fromthe blood sample, after which GAG contained in the extract is measured.No particular limitation is imposed on the method For extractinghydrophilic components; for example, a droplet of a blood sample isadded onto commercial filter paper, and hydrophilic components areextracted from the filter paper. Extraction of the hydrophiliccomponents from the filter paper may be Conveniently performed bysoaking, in an aqueous solution, the filter paper bearing the droplet ofthe blood sample.

No particular limitation is imposed on the subjects from which bodyfluids are collected, so long as they are individuals having chances ofsuffering from lysosomal storage diseases. Preferably, such individualsare mammals, and more preferably the mammals are humans. In particular,humans ranging in age from just-delivered newborns to about 6-monthinfants are preferred.

The “GAG of a single species” to be used in the method of the presentinvention should be understood in its literal meaning; i.e., one speciesof GAG. There may be cases where such a single species of GAG is linkedwith other components to thereby form a complex, and in such cases, theexpression “GAG of a single species” is used to mean that a singlespecies of GAG is present in the complex. For example, when GAG is boundto a protein and forms proteoglycan, “GAG of a single species” refers tothe case where a single species of GAG is present in the proteoglycan.

Examples of “GAG” include KS, HS, CS, DS (which is sometimes calledchondroitin sulfate B), Hep, and HA. Preferably, GAG has a sulfategroup. In particular, GAG is preferably KS, HS, CS, or DS, morepreferably KS or HS, most preferably KS.

Examples of “proteoglycan” formed up of GAG and protein attached theretoinclude, but are not limited to, decorin (DS is attached to the coreprotein), aggrecan (CS and KS are attached to the core protein),versican (DS is attached to the core protein), keratocan (KS is attachedto the core protein), syndecan (CS and HS are attached to the coreprotein), and perlecan (HS is attached to the core protein).

No particular limitation is imposed on the method for the “measurement”of any single species of these Was contained in a specimen, so long asthe method enables detection of the single species of GAG. As usedherein, the word “measurement” (or “assay”) encompasses not onlyquantitative detection of GAG but also qualitative detection (i.e.,detection of presence or absence of GAG).

Measurement of GAG of a single species (or in other words, measurementof a certain GAG species) may be performed through any of the followingmethods:

i) a method employing a polypeptide capable of specifically binding tothe GAG;

ii) a method for analysis using various chromatography. The method usingchromatography is not particularly limited, and includes variouschromatography methods such as a gas chromatography method and a liquidchromatography. Examples include a method in which a degradation enzymecapable of specifically reacting with a certain species of GAG is causedto react with the GAG contained in a specimen, to thereby obtain adegradation product (disaccharide), and through high performance liquidchromatography (HPLC) the elution time of the product from the ionexchange column is analyzed (disaccharide analysis);

iii) a method in which a degradation enzyme capable of specificallyreacting with a certain species of GAG is caused to react with the GAGcontained in a specimen, and the presence or absence, as well as thedegree, of degradation of the GAG is determined by use of a dye capableof reacting with the GAG;

iv) a method in which a degradation enzyme capable of specificallyreacting with a certain species of GAG is caused to react with the GAGcontained in a specimen, and the presence or absence, as well as thedegree, of degradation of the GAG is determined by use of a polypeptidecapable of specifically binding to the GAG. The method iv) is alsodescribed hereinbelow in Example 1; and

v) a method for analysis using a mass spectrometry. The method using amass spectrometer is not particularly limited, and includes various massspectrometry methods such as a tandem mass spectrometry method (MS/MS)and a MALDI/TOFMS method.

Of these methods, those employing a polypeptide capable of specificallybinding to GAG are preferred.

Preferably, the “polypeptide” is an antibody or a polypeptide having anantigen-binding site (Fab) of an antibody. The “polypeptide having anFab of an antibody” may be a fragment containing an Fab of an antibody.The Fab-containing fragment may be produced by treating an antibody witha protease (such as plasmin, pepsin, papain or the like) that does notdegrade Fab. Examples of the Fab-containing fragment also include Fabc,(Fab′)₂ and the like, in addition to Fab.

The “polypeptide having an Fab of an antibody” may be a chimera antibodyhaving an Fab of interest. When the nucleotide sequence of a gene codingfor the antibody or the amino acid sequence of the antibody isdetermined, there can be genetically produced a chimera antibody havingan Fab of interest or a fragment containing an Fab of interest.

The “polypeptide” is preferably purified in advance. When thepolypeptide is an “antibody” and the immunoglobulin class thereof isIgG, the polypeptide can be purified by means of affinity chromatographymaking use of protein A or Protein G. When the immunoglobulin class ofan antibody is IgM, the polypeptide is purified by means of gelfiltration column chromatography.

No particular limitation is imposed on the “antibody” employed in thepresent invention, so long as it is capable of specifically binding to aGAG of a single species and either of monoclonal antibodies andpolyclonal antibodies may be used. From the viewpoints of specificity,homogeneity, reproducibility, and productivity in sustainable abundance,monoclonal antibodies are preferred.

The “antibody” may be produced by a known method (for example, foranti-KS antibody, see J. Biol. Chem., 258, 8848-8854 (1983); and foranti-CS antibody, see J. Biol. Chem., 262, 4146-4152 (1987)).Alternatively, the “antibody” may be produced by following either of thebelow-described general methods.

1) Method for Producing a Polyclonal Antibody

An antigen is administered to animals for immunization, such as mice,rats, guinea pigs, rabbits, goat, sheep, horses, pigs, dogs, cats, orchickens under the skin (subcutaneously), into the abdominal cavity(intraperitoneally), or in a footpad.

Use of an adjuvant during immunization of animals is preferred, sinceadjuvants activate antibody-producing cells. When the animals receiveboosters in a usual manner two to three weeks following the initialimmunization, antisera of high titer can be obtained. About one weekfollowing the final immunization, blood is collected and serum isseparated. The serum is treated with heat to thereby deactivatecomplements. Immunoglobulin fractions may be purified through aconventional purification method employed for antibodies.

2) Method for Producing a Monoclonal Antibody

A monoclonal antibody may be prepared by the method of Kohler andMilstein (Nature, 256, 495-497 (1975)).

For example, an antigen is administered to animals for immunization,such as mice, rats, guinea pigs, rabbits, goat, sheep, horses, pigs,dogs, cats, or chickens intraperitoneally, subcutaneously, or in afootpad.

From the immunized animals, spleen cells, lymphocytes, peripheral blood,etc. are collected, and subjected to cell fusion with myeloma cells(which are of tumor cell line), to thereby prepare hybridomas. Themyeloma cells to be used in cell fusion may be obtained from variouscell lines of mammals. Preferably, cell lines from animals of the samespecies as the immunized animals are employed. Also preferably, themyeloma cells bear a marker for discernment, after cell fusion, betweenunfused cells and fused cells, to thereby enable growth of onlyhybridomas, while preventing survival of unfused myeloma cells. Also, inorder to facilitate collection of antibodies of interest from a culturesupernatant of hybridoma, the myeloma cells are preferably of a cellline which does not secrete inherent immunoglobulins.

The obtained hybridomas are continuously grown, and subsequently,hybridoma cell lines which continuously produce antibodies capable ofspecifically binding to antigens are selected through screening.

The thus-selected hybridoma cell line is cultured by use of a suitablemedium, to thereby obtain monoclonal antibodies in the medium. It isalso possible to mass-produce monoclonal antibodies by culturing saidhybridoma cell line in a living body; e.g., in the abdominal cavity of amouse, and then separating the hybridoma cell line from the ascites. Thethus-obtained monoclonal antibodies may be purified through aconventional purification method employed for antibodies.

Although no particular limitation is imposed on the immunoglobulin classof the antibodies, IgG is preferred. Antibodies whose immunoglobulinclass is IgG can be obtained through screening by use of an anti-IgGantibody.

The definition of “GAG of a single species” has already been providedhereinabove. Thus, the “polypeptide capable of specifically binding toGAG” is preferably a polypeptide capable of specifically binding to aGAG having a sulfate group; more preferably a polypeptide capable ofspecifically binding to KS, HS, CS, or DS; even more preferably apolypeptide capable of specifically binding to KS or HS; most preferablya polypeptide capable of specifically binding to KS.

The “polypeptide capable of specifically binding to GAG” may be, but isnot limited to, the commercial products described below. Thedescriptions in parentheses indicate animals from which theimmunoglobulins are obtained and the immunoglobulin classes.

Polypeptide Capable of Specifically Binding to KS:

Anti-KS antibody “5D4” (mouse, IgG1). “5D4” is a monoclonal antibodywhich specifically binds to KS.

Polypeptide Capable of Specifically Binding to HS:

Anti-HS antibodies “HepSS-1” (mouse, IgM), “F58-10E4” (mouse, IgM),“HK-249” (mouse, IgM), “F69-3G10” (mouse, IgG2b), “JM403” (mouse, IgM(Diabetologia, 37(3), 313-320 (1994))). These are all monoclonalantibodies which specifically bind to HS.

Polypeptide Capable of Specifically Binding to DS and CS:

Anti-CS antibodies “CS-56” (mouse, IgM), “MO-225” (mouse, IgM), “MC21C”(mouse, IgM), “LY111” (mouse, IgM), “1-B-5” (mouse, IgG1), “2-B-6”(mouse, IgG1), “3-B-3” (mouse, IgM), “2H6” (mouse, IgM), and “473”(mouse, IgA). These are all monoclonal antibodies which specificallybind to HS.

The above antibodies are already known and, except for JM403, arecommercially available from Seikagaku Corporation (Tokyo). Therefore,persona having ordinary skill in the art can prepare or procure themwithout difficulty.

Example assay methods for “GAG of a single species” by use of apolypeptide capable of specifically binding to GAG include, but are notlimited to, the following.

i) A specimen is brought into contact with a solid phase to which afirst polypeptide is immobilized, followed by addition of a secondpolypeptide for contact thereto, to thereby form a sandwich-likecomplex, and the thus-formed complex is detected (a so-called sandwichassay).

ii) In the presence of three components; that is, GAG-containingmolecules which is immobilized onto a solid phase, a specimen, and apolypeptide (wherein the specimen and the polypeptide may be broughtinto contact in advance), the GAG-containing molecules which isimmobilized and GAG-containing molecules contained in the specimen areallowed to competitively react with the polypeptide, and subsequently,the amount of polypeptide bound to the solid phase is detected, tothereby obtain the amount of GAG contained in the specimen (a so-calledinhibition assay).

iii) A specimen is brought into contact with fine particles to whichpolypeptide molecules is immobilized, followed by addition of a secondpolypeptide for contact thereto, to thereby form aggregates ofparticles, and the thus-formed aggregates (or precipitates) are detected(a so-called agglutination assay).

Preferably, the present method is carried out through a sandwich assay.That is, the present method preferably comprises the following steps:

(1) a step for forming a sandwich-like complex by bringing “a solidphase to which a first polypeptide capable of specifically binding to aGAG-containing molecule is immobilized”, “a specimen”, and “a secondpolypeptide capable of specifically binding to a GAG-containingmolecule” into contact with one another, the sandwich-like complex beingconstituted by “said first polypeptide immobilized onto the solidphase—GAG-containing molecule in the specimen—second polypeptide”; and

(2) a step for detecting the sandwich-like complex formed in step (1).

In step (1) above, the three substances; i.e., “a solid phase to which afirst polypeptide capable of specifically binding to a GAG-containingmolecule is immobilized”, “a specimen”, and “a second polypeptidecapable of specifically binding to a GAG-containing molecule” may bebrought into contact simultaneously. Alternatively, the former twosubstances may first be brought into contact with each other, followedby addition of the third substance for contact; or the latter twosubstances may first be brought into contact with each other, followedby addition of the first substance for contact. Preferably, according tothe method of the present invention, the former two substances are firstbrought into contact with each other, followed by addition of the thirdsubstance for contact. Thus, more preferably, the assay comprises thefollowing steps (1), (2), and (3):

(1) a step for forming a complex by bringing “a solid phase to which afirst polypeptide capable of specifically binding to a GAG-containingmolecule is immobilized” into contact with “a specimen”, the complexbeing constituted by “first polypeptide immobilised onto the solidphase—GAG-containing molecule in the specimen”;

(2) a step for forming a sandwich-like complex by bringing theabove-described solid phase into contact with “a second polypeptidecapable of specifically binding to a GAG-containing molecule”, thesandwich-like complex being constituted by “said first polypeptideimmobilized onto the solid phase—GAG-containing molecule in thespecimen—second polypeptide”; and

(3) a step for detecting the sandwich-like complex formed in step (2).

Hereafter, this method will be explained in detail for every process.

Step (1):

In step (1), “a solid phase to which a first polypeptide capable ofspecifically binding to a GAG-containing molecule is immobilized” isbrought into contact with “a specimen”, to thereby form a complexconstituted by “first polypeptide immobilized onto the solidphase—GAG-containing molecule in the specimen.”

(1)-1 First Polypeptide Capable of Specifically Binding to aGAG-Containing Molecule

As used herein, the term “GAG-containing molecule” refers to anymolecule that contains GAG as a constituent thereof. Examples of such amolecule include a GAG molecule per se (i.e., containing no otherconstituents), and a proteoglycan molecule.

The “first polypeptide” in this context may be the same as or differentfrom the below-described “second polypeptide.” However, in any case, atleast one of the two must be a polypeptide capable of specificallybinding to GAG (i.e., a GAG molecule per se).

The definition of “polypeptide capable of specifically binding to GAG(i.e., a GAG molecule per se)” has already been provided hereinabove. Anexample of a “polypeptide capable of specifically binding to aGAG-containing molecule (other than the GAG molecule per se)” is anantibody capable of specifically binding to the core protein ofproteoglycan. Examples of such an antibody include, but are not limitedto, “6-B-6” (mouse, IgG1) (anti-decorin antibody), “2-B-1” (mouse, IgG1)(anti-versican antibody), “HK-102” (mouse, IgG2b) (anti-perlecanantibody), “1-G-2” (mouse, IgG1) (anti-neurocan antibody), and “6-B-4”(mouse, IgM) (anti-phosphacan antibody). In the above description, theanimals from which the immunoglobulins are derived and immunoglobulinclasses are provided in parentheses.

The above are all monoclonal antibodies which are capable of binding tothe core protein of proteoglycans. These antibodies are commerciallyavailable from Seikagaku Corporation (Tokyo).

When blood is used as a specimen, GAGs contained in the blood specimenare considered to be present in the form of proteoglycans. Therefore,the aforementioned “polypeptide capable of specifically binding to aGAG-containing molecule (other than the GAG molecule per se)” may beemployed to serve as either one of the first polypeptide and a secondpolypeptide, which will be described hereinbelow. In this case, however,the other polypeptide must be a “polypeptide capable of specificallybinding to GAG (i.e., a GAG molecule per se).”

(1)-2 Solid Phase

No particular limitation is imposed on the solid phase to which thefirst polypeptide is to be immobilised, so long as the solid phase iscapable of immobilising the polypeptide and is insoluble in water,specimen, or reaction mixture of the assay. The solid phase may take avariety of forms, such as plates (e.g., wells of microplates), tubes,beads, membranes, gels, and micro-spherical solid carriers (gelatinparticles, kaolin particles, or synthetic polymer particles such aslatex). From the viewpoints of accurate quantitative evaluation andconvenience in use, microplates are preferred.

Examples of the material that constitutes the solid phase includepolystyrene, polypropylene, poly(vinyl chloride), nitrocellulose, Nylon,polyacrylamide, Teflon, polyallomer, polyethylene, glass, and agarose.Among these materials, polystyrene is preferred, and thus, plates madeof polystyrene are preferred.

In order to immobilize the first polypeptide onto any of these solidphases, a conventional method for preparing immobilized enzymes may beapplied, and such a method includes, for example, physical adsorption,covalent bonding, or entrapment (“Immobilized Enzymes” published byKodansha, 1975, pp. 9-75).

In particular, physical adsorption is preferred from the viewpoints ofconvenience in procedure and prevalence in use.

A specific example of physical adsorption is described below. Thisexample is drawn to a case where the first polypeptide is anti-KSantibody.

Anti-KS antibodies are dissolved in a buffer (e.g., phosphate buffer,phosphate buffered saline (PBS), or carbonate buffer; pH 7 to 9), andthe solution is added onto a solid phase (such as a microplate),followed by storage for 1 to 2 hours at about 37° C. or overnight atabout 4° C., to thereby immobilise the antibodies.

The surface of the solid phase to which the first polypeptide isimmobilised may have portions bearing no peptide, and whenGAG-containing molecules contained in the specimen adhere in annon-specific manner, accurate assay results may fail to be obtained. Toprevent this, preferably, a blocking substance is added before thespecimen is brought into contact with the solid phase, so as to coverthe portions to which the first polypeptide has not yet beenimmobilized. Examples of such a blocking substance include serumalbumin, casein, skim milk, gelatin, and Pluronic, and commercialproducts sold as such may also be employed.

In an exemplary blocking procedure, a blocking substance is added,followed by standing for 30 minutes to 2 hours at 37° C. or for 1 to 2hours at room temperature (15-25° C.).

(1)-3 Specimen

Relevant descriptions provided hereinabove apply, and thereforerepetition is omitted.

(1)-4 Contact between Solid Phase and Specimen

No particular limitation is imposed on the manner in which the solidphase is brought into contact with the specimen, so long as the firstpolypeptide molecules immobilized onto the solid phase and theGAG-containing molecules contained in the specimen are under conditionsallowing contact therebetween. For example, the specimen may be addedonto the solid phase, or vice versa, for achieving contact therebetween.Alternatively, the two may be simultaneously added into a containerwhich is provided separately. These are only examples, and contactbetween solid phase and specimen may be appropriately determined bypersons having ordinary skill in the art in accordance with the shape,material, etc. of the solid phase.

When contact between the two has been established, preferably, the firstpolypeptide and GAG-containing molecules contained in the specimen areallowed to react at 4 to 37° C., more preferably at 37° C., for about 1hour, so as to attain sufficient, complete bonding therebetween.

After completion of the above reaction, solid and liquid phases areseparated from each other. Preferably, non-specific adsorbents orunreacted components remaining in the specimen are removed by washingthe surface of the solid phase with a washing solution as desired.

Examples of preferred washing solutions include buffers to whichnonionic surfactants (such as those of the Tween series) areincorporated, and specifically, mention may be given of phosphatebuffer, PHS, and Tris HCl buffer.

When the specimen is brought into contact with the solid phase to whichthe first polypeptide is immobilized, a ° ampler of “first polypeptideimmobilized onto the solid phase—GAG-containing molecule” is formed.

Step (2):

In step (2), a second polypeptide capable of specifically binding to aGAG-containing molecule is brought into contact with the above-describedsolid phase that has undergone step (1), to thereby form a sandwich-likecomplex of “the first polypeptide immobilised onto the solidphase—GAG-containing molecule in the specimen—second polypeptide.”

(2)-1 Second Polypeptide Capable of Specifically Binding to aGAG-Containing Molecule

The descriptions provided hereinabove for the aforementioned “firstpolypeptide” also apply to the second polypeptide.

Preferably, the second polypeptide is labeled with, or can be labeledwith, a labeling substance, so as to facilitate detection thereof. Noparticular limitation is imposed on the labeling substance which may beemployed for labeling, so long as it can be ordinarily used for labelingproteins. Examples of such labeling substance include enzymes (such asperoxidase, alkaline phosphatase, β-galactosidase, luciferase,acetylcholinesterase, and glucose oxidase), radioisotopes (such as ¹²⁵I,¹³¹I, and ³H); fluorochromes (such as fluorescein isothiocyanate (FITC),7-amino-4-methylcoumarin-3-acetate (AMCA), dichlorotriazinylaminofluorescein (DTAF), tetramethylrhodamine isothiocyanate (TRITC),Lissamine Rhodamine B, Texas Red, Phycoerythrin (PE), umbelliferone,europium, phycocyanin, Tricolor, and crania); chemiluminescencesubstances (such as luminol); haptens (such as dinitrofluorobenzene,adenosine monophosphate (AMP), and 2,4-dinitroaniline); one component ofany of specific binding pairs (such as biotin and an avidin (e.g.,streptavidin), lectin and sugar chain, agonist and receptor therefor,heparin and antithrombin III (ATIII), and polysaccharide and a bindingprotein therefor (e.g., hyaluronic acid and hyaluronic-acid-bindingprotein (HABP)).

Of such exemplified labeling substances, one component of any ofspecific binding pairs is preferred, with either biotin or an avidinbeing more preferred. In particular, biotin is preferred.

The method for labeling the second polypeptide with a labeling substancemay be performed through any known method suited for the substance ofinterest. For example, when the labeling substance is an enzyme, any ofthe following methods may be appropriately employed: glutaraldehydemethod, periodate cross-linking method, maleimide cross-linking method,carbodiimide method, and activated ester method. When the labelingsubstance is a radioisotope, the chloramine T method or thelactoperoxidase method (see Zoku-Seikagaku Jikken Koza 2 “ProteinChemistry (the last volume)” published by Tokyo Kagaku Dojin, 1987) maybe appropriately employed. For example, when biotin is employed as alabeling substance, there may be used a method in which anN-hydroxysuccinimide ester derivative or hydrazide derivative of biotin(see Avidin-Biotin Chemistry: A Handbook, PP. 57-63, Pierce ChemicalCompany, published in 1994).

Preferably, the second polypeptide is labeled with a labeling substancein advance.

(2)-2 Contact between Solid Phase that has undergone Step (1) and SecondPolypeptide

This step may be performed in a manner similar to that described in(1)-4 above. Also, similar to the case of step (1)-4, after completionof reaction, solid and liquid phases are separated from each other, andpreferably, in accordance with needs, non-specific adsorbents and theunreacted components remaining in the specimen are removed throughwashing of the surface of the solid phase. Moreover, employable washingsolutions are the same as described in connection with step (1)-4.

When the aforementioned solid phase that has undergone step (1) (i.e.,the solid phase bearing a complex of “first polypeptide immobilized ontothe solid phase—GAG-containing molecule in the specimen”) is broughtinto contact with the second polypeptide capable of specifically bindingto a GAG-containing molecule, a sandwich-like complex of “the firstpolypeptide immobilized onto the solid phase—GAG-containing molecule inthe specimen—second polypeptide” is formed.

Step (3):

In step (3), the sandwich-like complex formed in step (2) is detected.

No particular limitation is imposed on the method for detecting thesandwich-like complex. For example, when the second polypeptide islabeled with a labeling substance, the complex can be detected bydetecting the labeling substance.

Labeling substances may be detected by an appropriate method selectedfrom among known methods established in accordance with the type oflabeling substances. For example, when one component (e.g., biotin) of acertain specific binding pair is employed as a labeling substance, anenzyme (such as peroxidase) to which another component (e.g.,streptavidin) capable of specifically binding to the first component isadded, to thereby cause formation of a specific binding pair.Subsequently, a substrate (for example, hydrogen peroxide (in the casewhere the enzyme is peroxidase)) for the enzyme employed and achromogenic substance (such as 3,3′,5,5′-tetramethylbenzidine (TMB) ordiaminobenzidine) is added, and color developed and assumed by theproduct of the enzymatic reaction is determined through absorptiometry,to thereby detect the labeled substance.

When a radioisotope, a fluorochrome, or a chemoluminescence substanceserves as a labeling substance, radioactivity count, fluorescenceintensity, fluorescence polarization, or luminescence intensity may bemeasured.

Through detection of such a labeling substance, the sandwich-likecomplex can be detected, attaining measurement of GAG of a singlespecies (that is, a certain species of GAG) contained in the specimen.Because this method is a sandwich format, detection of a large amount oflabeling substance indicates a commensurately large amount of a sandwichcomplex; in other words, presence of a large amount of GAG of a singlespecies in the specimen.

When a qualitative assay of GAG (detection of the presence or absence ofGAG) is desired, the results (positive or negative) regarding detectionof the labeling substance is directly employed as the assay results forGAG.

When a quantitative assay of GAG (such as measurement of the GAGconcentration) is desired, the absorbance value, radioactivity count,fluorescent intensity, or luminescence intensity may be directlyemployed as an index for the GAG content. Moreover, by use of standardGAGs of known concentrations, there may be prepared in advancecalibration curves or correlation equations regarding the relationbetween GAG concentration and results of detection (e.g., absorbance) onstandard substances, and the GAG concentration in the specimen may bedetermined therefrom. When a urine sample is employed as a specimen, thecalculated GAG concentration may be corrected with reference to theconcentrations of other substances (such as urine creatinine) containedin urine.

Another preferred method for the present invention is the inhibitionmethod. That is, the method of the invention is preferably preformedthrough a method comprising the following steps (1) and (2):

(1) a step for forming first and second complexes by bringing “a thirdpolypeptide capable of specifically binding to a GAG-containingmolecule”, “a specimen”, and “a solid phase to which a GAG-containingmolecule is immobilized” into contact with one another, the firstcomplex being constituted by “GAG-containing molecule immobilized onto asolid phase—third polypeptide” and the second complex being constitutedby “GAG-containing molecule in the specimen—third polypeptide”; and

(2) a step for detecting at least one of the complexes formed in step(1), the first complex being “GAG-containing molecule immobilized onto asolid phase—third polypeptide” and the second complex being“GAG-containing molecule in the specimen—third polypeptide.”

In step (1), the three substances; i.e., “a third polypeptide capable ofspecifically binding to a GAG-containing molecule”, “a specimen”, and “asolid phase to which a GAG-containing molecule is immobilized” may bebrought into contact simultaneously. Alternatively, the former twosubstances may first be brought into contact with each other, followedby addition of the third substance for contact; or the latter twosubstances may first be brought into contact with each other, followedby addition of the first substance for contact. Preferably, according tothe method of the present invention, the former two substances are firstbrought into contact with each other, followed by addition of the thirdsubstance for contact.

In step (2), of the first and second complexes, only the first complex(i.e., “GAG-containing molecule immobilized onto a solid phase—thirdpolypeptide”) or the second complex (i.e., “GAG-containing molecule inthe specimen—third polypeptide”), or both, may be detected. According tothe present method, detection of only the first complex is preferred.

That is, the method of the present invention preferably comprises thefollowing steps (1) to (3):

(1) a step for forming a first complex by bringing “a third polypeptidecapable of specifically binding to a GAG-containing molecule” and “aspecimen” into contact, the first complex being constituted by “thirdpolypeptide—GAG-containing molecule in the specimen”;

(2) a step for forming a second complex by bringing “the solid phase towhich a GAG-containing molecule is immobilized” into contact with amixture resulting from step (1); i.e., a mixture containing “the firstcomplex” and “a third polypeptide that has not participated in formationof the first complex”, the second complex being constituted by“GAG-containing molecule immobilized onto the solid phase—thirdpolypeptide”; and

(3) a step for detecting the second complex formed in step (2).

Detection of the second complex is preferably carried out by use of afourth polypeptide capable of specifically binding to the thirdpolypeptide and having been labeled with, or being capable of beinglabeled with, a labeling substance.

Respective steps of the assay will next be described.

Step (1):

In step (1), a first complex is formed by bringing “a third polypeptidecapable of specifically binding to a GAG-containing molecule” and “aspecimen” into contact, the first complex being constituted by “thirdpolypeptide—GAG-containing molecule in the specimen.”

The description provided for the “third polypeptide capable ofspecifically binding to a GAG-containing molecule” also applies to theaforementioned “polypeptide capable of specifically binding to GAG(i.e., a GAG molecule itself).” Also, the specimen employed in this stepis the same as that described above. No particular limitation is imposedon the manner of contact between the third polypeptide and the specimen,so long as the third polypeptide molecule can be brought into contactwith a GAG-containing molecule contained in the specimen.

When the third polypeptide is brought into contact with the specimen, afirst complex; i.e., a “third polypeptide—GAG-containing molecule in thespecimen” is formed. As a result, step (1) can yield a mixturecontaining “the first complex” and “a third polypeptide that has notparticipated in formation of the first complex.”

Step (2):

In step (2), the solid phase to which the GAG-containing molecule isimmobilized is brought into contact with the mixture obtained from step(1); i.e., a mixture containing “the first complex” and “the thirdpolypeptide that has not participated in formation of the firstcomplex”, to thereby form a complex of “GAG-containing moleculeimmobilized onto a solid phase—third polypeptide.”

No particular limitation is imposed on the solid phase to whichGAG-containing molecules are to be immobilized, so long as the solidphase is capable of immobilizing GAG-containing molecules and isinsoluble in water, specimen, or reaction mixture of the assay. Forother materials, see the descriptions provided hereinabove.

Also, the GAG-containing molecules capable of being immobilized onto thesolid phase are the same as those described hereinabove. No particularlimitation is imposed on the GAG-containing molecules capable of beingimmobilized onto the solid phase, so long as they have a site (or aportion) to which the third polypeptide can be specifically bound. Thatis, the GAG-containing molecule encompasses not only the GAG-containingmolecule itself but also a fragment obtained through treatment of theGAG-containing molecule with a GAG-specific degradation enzyme.

GAG-containing molecules may be immobilized onto a solid phase throughany conventional method, such as physical adsorption or covalentbonding. In particular, physical adsorption is preferred, from theviewpoints of convenience in procedure and prevalence in use.

The solid phase to which the GAG-containing molecules is immobilized isbrought into contact with the mixture obtained in step (1) in a mannersimilar to that described above. Similar to the aforementioned case,preferably, the solid phase and liquid phase are separated from eachother after completion of reaction, and in addition, in accordance withneeds, non-specific adsorbents and the unreacted components remaining inthe specimen are removed through washing of the surface of the solidphase with a washing solution. The washing solution which may beemployed is as described in the foregoing.

By bringing the solid phase to which GAG-containing molecules isimmobilized into contact with the mixture obtained from step (1), the“third polypeptides that have not participated in formation of the firstcomplexes” are specifically bound to the GAG-containing molecules, tothereby form the complexes of the “GAG-containing molecules immobilizedonto the solid phase—third polypeptides.”

Step (3);

In step (3), the complexes formed in step (2) are detected.

No particular limitation is imposed on the method for detecting thecomplexes. However, detection is preferably performed by use of “fourthpolypeptides capable of specifically binding to third polypeptides andhaving been labeled with, or being capable of being labeled with, alabeling substance.”

No particular limitation is imposed on the “fourth polypeptides capableof specifically binding to third polypeptides” so long as the fourthpolypeptides can specifically bind to the third polypeptides. When thethird polypeptides are antibodies (immunoglobulins), the fourthpolypeptides may be antibodies which can specifically bind to suchimmunoglobulins in accordance with the animals from which theimmunoglobulins are derived or the class of the immunoglobulins. Forexample, when the third polypeptide is an immunoglobulin derived frommouse (mouse IgG1), en anti-mouse IgG1 antibody may nerve as the fourthpolypeptide.

The descriptions provided for the previously mentioned labelingsubstance are also applicable to the labeling substance employable forlabeling the fourth polypeptides. Preferably, the labeling substance isan enzyme (peroxidase, alkaline phosphatase, β-galactosidase,luciferase, acetylcholinesterase, glucose oxides, etc.), with peroxidasebeing more preferred.

Since the method for labeling the fourth polypeptides with a labelingsubstance and the method for detecting the labeling substance areperformed in manners similar to those described hereinabove,descriptions therefor are omitted for the sake of simplicity. However,since the employed method is the inhibition method, detection of a largeamount of the labeling substance should be interpreted such that theamount of the “third polypeptides that have not participated information of ‘third polypeptide—GAG-containing molecule in the specimen’complexes” is commensurably large (that is, the amount of the complexesbeing commensurably small); in other words, the amount of the GAG of asingle species contained in the specimen is small.

In the present invention, no particular limitation is imposed on the“lysosomal storage diseases” which are to be detected by the presentmethod and are correlated with assay results of GAGs, so long as the“lysosomal storage diseases” are diseases recognized as such in the art.Preferably, the “lysosomal storage diseases” are at least one diseaseselected from mucopolysaccharidoses, mucolipidoses, GM1 gangliosidoses,fucosidoses, galactosialidoses, metachromatic leukodystropy,Niemann-Pick diseases, Tay-Sachs disease, Sandhoff disease, GM2gangliosidoses, Krabbe disease, Fabry disease, Gaucher diseases,glycogen storage diseases and lipofuscinoses.

In the present invention, no particular limitation is imposed on the“mucopolysaccharidoses” which are to be detected by the present methodand are correlated with assay results of GAGs, so long as the“mucopolysaccharidoses” are diseases recognized as such in the art.Preferably, the “mucopolysaccharidoses” are a class ofmucopolysaccharidosis types I, II, III, IV, VI, and VII.

In the present invention, no particular limitation is imposed on the“mucolipidoses”, “GM1 gangliosidoses”, “fucosidoses”,“galactosialidoses”, “metachromatic leukodystropy”, “Niemann-Pickdiseases”, “Tay-Sachs disease”, “Sandhoff disease”, “GM2gangliosidoses”, “Krabbe disease”, “Fabry disease”, “Gaucher diseases”,“glycogen storage diseases” and “lipofuscinoses” so long as they arediseases recognized as such in the art. Preferably, the “mucolipidoses”are mucolipidosis types II or III. Preferably, the “Niemann-Pickdiseases” are Niemann-Pick disease types B or C. Preferably, the“Gaucher diseases” are Gaucher disease types I or III. Preferably, the“glycogen storage diseases” are glycogen storage disease types 1 or 2.

The step in which lysosomal storage diseases are correlated with assayresults of GAGs may be performed as follows.

As described above, a specimen from an animal of lysosomal storagediseases shows a significantly high GAG level. Accordingly, when themeasurement of GAG of a single species (GAG level) is higher than that(GAG level) of healthy animals (animals of non-lysosomal storagediseases), the measurement can be correlated to “affirmation oflysosomal storage diseases” or “high risk of lysosomal storage diseases”

When the measurement of GAG of a single species (GAG level) is lowerthan that (GAG level) of healthy an the measurement can be correlated to“free of lysosomal storage diseases” or “low risk of lysosomal storagediseases”

The correlation between the identified GAG level and lysosomal storagediseases encompasses not only that for predicting the “presence orabsence of the risk of lysosomal storage diseases” but also that forpredicting the severity or progress of lysosomal storage diseases. Forexample, if the GAG level of a specimen obtained from a certainindividual is periodically measured and shows a tendency of increase inthe GAG level, such a tendency may be correlated to “progressivelysosomal storage diseases” or “high risk of lysosomal storage diseasesprogressing.” On the other hand, when the results show a tendency ofdecrease in the GAG level, such a tendency may be correlated to“lysosomal storage diseases ameliorating” or “high possibility oflysosomal storage diseases ameliorating.” Also, when no changes in theGAG level are observed, this can be correlated to “no changes in thestate of lysosomal storage diseases” or “high possibility of lysosomalstorage diseases neither progressing nor mitigating.”

The measurement (GAG level) which forms the basis for the correlationwith lysosomal storage diseases may be the GAG concentration obtained byuse of the aforementioned calibration curve or the correlationequations, or the GAG ratio with respect to the GAG level as determinedin a specimen from healthy animals.

According to the method of the present invention, the “GAG of a singlespecies” is preferably a GAG having a sulfate group. More preferably,the GAG having a sulfate group is KS, and simultaneously, the“mucopolysaccharidoses” to be detected are one or moremucopolysaccharidoses selected from mucopolysaccharidosis types I, II,III, VI, and VII. This is a particularly important feature of thepresent invention, since the finding that KS is secreted into bodyfluids of a subject suffering mucopolysaccharidosis type I, II, III, VIor VII has remained completely unknown until conception of the presentinvention. Among the above types, the “mucopolysaccharidoses” to bedetected are preferably one or more mucopolysaccharidoses selected fromtypes I, II, III and VI. The “GAG having a sulfate group” is preferablyKS, and simultaneously, the “mucolipidoses” to be detected are one ormore mucolipidoses selected from mucolipidoses types II and III.

In the present invention, the following case is also preferred: The GAGhaving a sulfate group is HS, and simultaneously, the“mucopolysaccharidoses” to be detected are one or moremucopolysaccharidoses selected from mucopolysaccharidosis types IV andVI. This is also a particularly important feature of the presentinvention, since the finding that HS is secreted into body fluids ofsubjects suffering mucopolysaccharidosis type IV or VI has remainedcompletely unknown until conception of the present invention.

In the present invention, the following case is also preferred: the GAGhaving a sulfate group is HS, and the lysosomal storage diseases are ofone or more diseases leukodystropy, Niemann-Pick diseases, Tay-Sachsdisease, Sandhoff disease, GM2 gangliosidoses, Krabbe disease, Fabrydisease, Gaucher diseases, glycogen storage diseases and lipofuscinoses.

In the present invention, the following case is also Preferred: The GAGhaving a sulfate group is CS, and simultaneously, the“mucopolysaccharidoses” to be detected are one or moremucopolysaccharidoses selected from mucopolysaccharidosis types I, II,III, IV, and VI. This is also a particularly important feature of thepresent invention, since the finding that CS is secreted into bodyfluids of subjects suffering mucopolysaccharidosis of the above typeshas remained completely unknown until conception of the presentinvention.

In the present invention, the following case is also preferred: The GAGhaving a sulfate group is DS, and simultaneously, the“mucopolysaccharidosis” to be detected are one or moremucopolysaccharidoses selected from mucopolysaccharidosis types III andIV. This is also a particularly important feature of the presentinvention, since the finding that DS is secreted into body fluids ofsubjects suffering mucopolysaccharidosis of the above types has remainedcompletely unknown until conception of the present invention.

Although the descriptions hereinabove have focused on the “detectionmethod” for lysosomal storage diseases, the method of the presentinvention is not necessarily limited only to such a method, and a“screening method” and a “diagnosis method” are also envisaged by thepresent invention.

<2> Kit of the Present Invention

The kit of the present invention includes the following components, andis intended to be used to detect at least one disease selected frommucopolysaccharidoses, mucolipidoses, GM1 gangliosidoses, fucosidoses,galactosialidoses, metachromatic leukodystropy, Niemann-Pick disease,Tay-Sachs disease, Sandhoff disease, GM2 gangliosidoses, Krabbe disease,Fabry disease, Gaucher disease, glycogen storage disease andlipofuscinoses on the basis of the measurement of GAG of a singlespecies in a specimen:

(A) a solid phase to which a first polypeptide capable of specificallybinding to a GAG-containing molecule is immobilized; and

(B) a second polypeptide capable of specifically binding to aGAG-containing molecule and having been labeled with, or being capableof being labeled with, a labeling substance.

In the following descriptions of the kit of the present invention, theterms “GAG of a single species”, “first polypeptide capable ofspecifically binding to a GAG-containing molecule”, “solid phase towhich a first polypeptide is immobilized”, “second polypeptide capableof specifically binding to a GAG-containing molecule”, “labelingsubstance”, method for labeling a polypeptide with a labeling substance,and target “mucopolysaccharidoses, etc.” to be detected all have thesame meanings as previously provided in section <1> Method of thePresent Invention. The present kit can be used to detect lysosomalstorage diseases through sandwich assay of GAGs.

Instead of the above components (A) and (B), the kit of the presentinvention may include the following components (A), (B), and (C):

(A) a solid phase to which a GAG-containing molecule is immobilized,

(B) a third polypeptide capable of specifically binding to aGAG-containing molecule, and

(C) a fourth polypeptide capable, of specifically binding to the thirdpolypeptide and having been labeled with, or being capable of beinglabeled with, a labeling substance.

In the following descriptions of the kit of the present invention, theterms “solid phase to which a GAG-containing molecule is immobilized”,“third polypeptide capable of specifically binding to a GAG-containingmolecule”, “fourth polypeptide capable of specifically binding to athird polypeptide”, “labeling substance”, method for labeling apolypeptide with a labeling substance, and target“mucopolysaccharidoses, etc.” to be detected all have the same meaningsas previously provided in section <1> Method of the Present Invention.The present kit can be used to detect mucopolysaccharidoses, etc.through the inhibition assay of GAGs.

Detection of mucopolysaccharidoses, etc. by use of any mode of the kitof the present invention can be achieved in accordance with thedescriptions provided in section <1> Method of the Present Invention.”

In the kit of the present invention, the “polypeptide” is preferably anantibody or a polypeptide having an antigen binding site of an antibody.

No particular limitation is imposed on the “mucopolysaccharidoses” whichare to be detected by the present kit, so long as the“mucopolysaccharidoses” are diseases recognized as such in the art.Preferably, the “mucopolysaccharidoses” axe a class ofmucopolysaccharidosis types I, II, III, IV, VI, and VII. In the samemanner, in the kit of the present invention, no particular limitation isimposed on the “mucolipidoses”, “GM1 gangliosidoses”, “fucosidoses”,“galactosialidoses”, “metachromatic leukodystropy”, “Niemann-Pickdiseases”, “Tay-Sachs disease”, “Sandhoff disease”, “GM2gangliosidoses”, “Krabbe disease”, “Fabry disease”, “Gaucher diseases”,“glycogen storage diseases” and “lipofuscinoses”, so long as they arediseases recognized as such in the art. Preferably, the “mucolipidoses”are mucolipidosis type II or III. Preferably, the “Niemann-Pickdiseases” are Niemann-Pick disease types B or C. Preferably, the“Gaucher diseases” are Gaucher disease types I or III. Preferably, the“glycogen storage diseases” are glycogen storage disease types 1 or 2.

Moreover, “GAG of a single species” in the kit of the present inventionis preferably a GAG having a sulfate group, and the GAG having a sulfategroup is preferably KS, HS, CS, or DS.

In the kit of the present invention, the “GAG having a sulfate group” ispreferably KS, and simultaneously, the “mucopolysaccharidoses” are oneor more mucopolysaccharidoses selected from mucopolysaccharidosis typesI, II, III, VI, and More preferably, the “mucopolysaccharidoses” to bedetected are one or more mucopolysaccharidoses selected frommucopolysaccharidosis types I, II, III, and VI. Also, the “GAG having asulfate group” is preferably KS, and simultaneously, the “mucolipidoses”are one or more mucolipidoses selected from mucolipidosis types II andIII.

Moreover, the following cases are also preferred: The “GAG having asulfate group” is HS, and simultaneously, the “mucopolysaccharidoses”are one or more mucopolysaccharidoses selected frommucopolysaccharidosis types IV and VI; the “GAG having a sulfate group”is HS, and the lysosomal storage diseases are of one or more diseasesselected from among mucolipidoses, metachromatic leukodystropy,Niemann-Pick diseases, Tay-Sachs disease, Sandhoff disease,gangliosidoses, Krabbe disease, Fabry disease, Gaucher diseases,glycogen storage diseases and lipofuscinoses; the “GAG having a sulfategroup” is CS, and simultaneously, the “mucopolysaccharidoses” are one ormore mucopolysaccharidoses selected from mucopolysaccharidosis types I,II, III, IV and VI; the “GAG having a sulfate group” is DS, andsimultaneously, the “mucopolysaccharidoses” are one or moremucopolysaccharidoses selected from mucopolysaccharidosis types III andIV. Note that these features also apply to the method of the presentinvention.

No particular limitation is imposed on the kit of the present invention,so long as the kit includes the above-described components. The kit mayfurther include standard GAG products of known concentration serving asstandard samples useful for drawing calibration curves or establishingcorrelation equations, detection reagents for labeling substances, andso on. In addition to these components, the kit may also include theaforementioned blocking substance, the aforementioned washing solution,a solution for diluting specimens, and a solution for stopping enzymaticreactions. Moreover, the kit may include a substance serving as apositive control (QC-control), which is used to maintain a certain assaylevel throughout the assay batches.

These components may be individually stored in separate containersforming a kit, which is subsequently used according to the method of thepresent invention.

Although the above description has focused on a “detection kit” formucopolysaccharidoses, needless to say, a “screening kit” and a“diagnosis kit” are also envisaged by the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the KS levels determined in human urinespecimens.

FIG. 2 is a graph showing the HS levels determined in human urinespecimens.

FIG. 3 is a graph showing the CS levels determined in human urinespecimens.

FIG. 4 is a graph showing the DS levels determined in human urinespecimens.

FIG. 5 is a graph showing the KS levels determined in human urinespecimens.

FIG. 6 is a graph showing the KS levels determined in human plasmaspecimens.

FIG. 7 is a graph showing the KS levels determined in human plasmaspecimens.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will next be described in more detail by way ofexamples, which should not be construed as limiting the inventionthereto.

Example 1

A sandwich assay was performed to detect mucopolysaccharidoses in humanurine samples.

(1) The Specimens, Reagents, Etc. Employed in Example 1 were as Follows.

Specimens and Standard Samples:

The specimens were human urine samples collected from patients sufferinghuman mucopolysaccharidosis types I, II, III, IV, or VI (1 subject each)and healthy humans (humans not suffering mucopolysaccharidosis; 2subjects).

The Standard GAGs Employed were as Follows:

* HS (derived from bovine kidney, produced by Seikagaku Corporation):

This KS is available from Seikagaku Corporation as reagent cataloguecode No. 400700, and has the following properties.

Nitrogen Content:

-   -   2.6 to 3.2% (measured by the method described in Z. Anal. Chem.,        22, 366 (1883))

Sulfur Content:

-   -   5.0 to 6.0% (measured by the method described in Mikrochim.        Acta., 123 (1955))

Uronic Acid Content:

-   -   28.0 to 30.0% (oxynol reaction)    -   36.0 to 40.0% (carbazole reaction)

Glucosamine Content:

-   -   30.0 to 35.0% (amino acid automatic analyzer)

Galactosamine Content:

-   -   <0.01%        * KS (keratan polysulfate; derived from shark cartilage;        produced by Seikagaku Corporation):

This KS is contained in a kit available from Seikagaku Corporation asreagent catalogue code No. 400610, and has the following properties.

Nitrogen Content:

-   -   2.58% (measured by the method described in Z. Anal. Chem., 22,        366 (1883))

Sulfur Content:

-   -   9.70% (measured by the method described in Mikrochim. Acta., 123        (1955))

Glucosamine Content:

-   -   23.51% (amino acid automatic analyzer)

Galactosamine Content:

-   -   0.11% (amino acid automatic analyzer)        Galactose content:    -   26.26% (Biochem. J., 50, 298 (1952))        * CS (chondroitin sulfate D; derived from shark cartilage;        produced by Seikagaku Corporation):

This CS is available from Seikagaku Corporation as reagent cataloguecode No. 400676, and has the following properties.

Nitrogen Content:

-   -   2.2 to 2.6% (measured by the method described in Z. Anal. Chem.,        22, 366 (1883))

Sulfur Content:

-   -   7.1 to 7.7% (measured by the method described in Mikrochim.        Acta., 123 (1955))

Galactosamine Content:

-   -   30 to 35% (amino acid automatic analyzer)

Glucuronic Acid Content:

-   -   32 to 35% (carbazole reaction)

Chondroitin sulfate D is a molecule in which “disaccharide units inwhich a glucuronic acid residue and an N-acetylgalactosamine residue arebound via β1,3-glycoside linkage” are continuously bound, and CScontaining, as a main constitution component, a disaccharide unitconsisting of “a glucuronic acid residue sulfated at the 2-position andan N-acetylgalactosamine residue sulfated at the 6-position.”

* Decorin (derived from bovine articular cartilage; produced by Sigma)

Antibodies:

“5D4” (produced by Seikagaku Corporation) was employed to serve as ananti-KS antibody to be immobilized onto a solid phase. “Biotinylated5D4” (produced by Seikagaku Corporation) was employed to serve as abiotinylated anti-KS antibody.

“F58-10E4” (produced by Seikagaku Corporation) was employed to serve asan anti-HS antibody to be immobilized onto a solid phase. “BiotinylatedF58-10E4” (produced by Seikagaku Corporation) was employed to serve as abiotinylated anti-HS antibody.

“LY111” (produced by Seikagaku Corporation) was employed to serve as ananti-CS antibody to be immobilised onto a solid phase. “BiotinylatedLY111” (produced by Seikagaku Corporation) was employed to serve as abiotinylated anti-CS antibody.

“6-8-6” (produced by Seikagaku Corporation) was employed to serve as anantibody for the core protein of proteoglycan (decorin).

Antibody-Immobilized Plates:

The antibody-immobilized plates (i.e., plates to which “5D4”,“F58-10E4”, “LY111”, or “6-B-6” antibodies were immobilized) wereprepared as described below.

Antibodies of each of the above species were dissolved in phosphatebuffered saline (PBS), to thereby adjust protein concentration to 20μg/ml. The solution was added to an immunoplate (MAXISORP; produced byNunc) in an amount of 50 μl/well, followed by incubation at 37° C. for1.5 hours.

After completion of incubation, the wells were washed twice with PBS,and the blocking substance (Immunoassay Stabilizer; produced by AppliedBiosystems) was added to each well in an amount of 200 μl/well.Subsequently, the wells were incubated at 37° C. for 1 hour.

The thus-prepared antibody-immobilized plates, when washed with awashing solution 3 times, are ready to use, and can also be used evenafter storage for several months following drying.

Reagents, etc.:

Washing solution: PBS containing 0.05% Tween 20

Specimen diluting solution: PBS(−) containing 1% bovine serum albumin(BSA)

(2) Detection of Mucopolysaccharidoses Through Measurement of KS

Each specimen was diluted with specimen diluting solution, to therebyprepare two sets of diluted specimens (0.5 ml each). To each specimensof a first set, keratanase II (2.5 mU, produced by SeikagakuCorporation) was added, whereas this enzyme was not added to thespecimens of the other set. Subsequently, all the specimens wereincubated at room temperature for 3 hours.

The incubated specimens (which may be called “assay specimens”) weresubjected to a GAG assay as described below, by use of the5D4-immobilized plate and biotinylated 5D4.

Each of the assay specimens was added to the wells of the respectiveantibody-immobilized plates described above in an amount of 50 μl/well,followed by incubation at 37° C. for 1 hour. Subsequently, washingsolution was added to the wells in an amount of 200 μl/well, and theplates were washed 4 times.

Biotinylated 5D4 was diluted with specimen diluting solution so as toattain a concentration of 0.5 μg/ml, and then was added to the wells ofeach antibody-immobilized plate in an amount of 50 μl/well, followed byincubation at 37° C. for 1 hour. Subsequently, washing solution wasadded to the wells in an amount of 200 μl/well, and the plates werewashed 4 times.

Avidin-peroxidase (produced by Vector) was subjected to 1,000-folddilution by use of specimen diluting solution, and then added to thewells of each antibody-immobilized plate in an amount of 50 μl/well,followed by incubation at 37° C. for 30 minutes. Subsequently, washingsolution was added to the wells in an amount of 200 μl/well, and theplates were washed 4 times.

A TMB solution (substrate, produced by Moss Inc.) was added to the wellsof each plate in an amount of 50 μl/well, then subjected to incubationat room temperature for 5 minutes. Next, 1 M HCl was added thereto in anamount of 50 μl/well, to thereby stop enzymatic reaction. The absorbanceat 450-630 nm was measured by use of an absorptiometer.

The measurement results of absorbance are shown in FIG. 1. The left barsof each bar set show the results obtained from the solutions that hadnot undergone any treatment with keratanase II, whereas the right barsshow the results obtained from the solutions that had undergonetreatment with keratanase II. In FIGS. 1 to 4, “Blank”, “MPS”, and“Normal” indicate the results corresponding to no specimen being added,mucopolysaccharidosis, and healthy subjects, respectively.

As shown in FIG. 1, urine samples collected from subjects suffering anyof mucopolysaccharidosis types I to VI were found to exhibitsignificantly high absorbance values as compared with the urine samplescollected from healthy subjects. Specimens treated with keratanase II(which specifically degrades KS) exhibited low absorbance values, whichare on similar levels. These results indicate that high absorbancevalues exhibited in urine samples from subjects suffering any ofmucopolysaccharidosis types I to VI are attributed to KS; in fact, inall the urine samples from the subjects suffering mucopolysaccharidosisof any type, the amount of KS was found to be significantly high.Particularly, high KS levels in urine samples of mucopolysaccharidosesistypes I, II, III, and VI are surprising, because such high data havenever been expected.

Thus, it has now been shown that mucopolysaccharidoses can be detectedby correlating the assay results of GAG of a single species (in thiscase, KS) contained in body fluid (urine) with mucopolysaccharidoses.

(3) Detection of Mucopolysaccharidoses Through Measurement of HS

Each specimen was diluted with specimen diluting solution, to therebyprepare two sets of diluted specimens (0.5 ml each). To each specimen ofa first set, heparitinase I (10 mU, produced by Seikagaku Corporation)was added, whereas this enzyme was not added to the specimens of theother set. Subsequently, all the specimens were incubated at roomtemperature for one hour. The standard samples (HS) also underwentsimilar treatment.

The incubated specimens (which may be called “assay specimens”) weresubjected to a GAG assay as described below, by use of anF58-10E4-immobilized plate and biotinylated F58-10E4.

Each of the assay specimens was added to the wells of the respectiveantibody-immobilized plates described above in an amount of 50 μl/well,followed by incubation at 37° C. for 1 hour. Subsequently, washingsolution was added to the wells in an amount of 200 μl/well, and theplates were washed 4 times.

Avidin-peroxidase (produced by Vector) diluted 1,000-fold with specimendiluting solution (4° C.) and biotinylated F58-10E4 diluted with aspecimen diluting solution (4° C.) to attain a concentration of 1.0μg/ml were added to the wells of each antibody-immobilized plate in anamount of 25 μl/well each, followed by incubation at 4° C. for 1 hour.Subsequently, washing solution was added to the wells in an amount of200 μl/well, and the plates were washed 4 times.

The steps from addition of TMB solution (substrate) up to measurement ofabsorbance were the same as those described in (2) above.

The measurement results of absorbance are shown in FIG. 2. The left barsof each bar set show the results obtained from the solutions that hadnot undergone any treatment with heparitinase I, whereas the right barsshow the results obtained from the solutions that had undergonetreatment with heparitinase I, “HS(+) control” indicates the resultsobtained from the cases where standard samples (HS) were employed.

As shown in FIG. 2, urine samples collected from subjects suffering anyof mucopolysaccharidosis types I to VI were found to exhibitsignificantly high absorbance values as compared with the urine samplescollected from healthy subjects. Specimens treated with heparitinase I(which specifically degrades HS) exhibit low absorbance values, whichare on similar levels. These results indicate that high absorbancevalues exhibited in urine samples from subjects suffering any ofmucopolysaccharidosis types I to VI are attributed to HS; in fact, inall the urine samples from the subjects suffering any ofmucopolysaccharidosis of any type, the amount of HS was found to besignificantly high. Particularly, high HS values in urine samples ofmucopolysaccharidosis types IV and VI are surprising, because such highdata have never been expected.

Thus, it has now been shown that mucopolysaccharidoses can be detectedby correlating the assay results of GAG of a single species (in thiscase, HS) contained in body fluid (urine) with mucopolysaccharidoses.

(4) Detection of Mucopolysaccharidoses Through Measurement of CS

Each specimen was diluted with specimen diluting solution, to therebyprepare diluted specimens (0.5 ml each).

The diluted specimens (which may be called “assay specimens”) weresubjected to a GAG assay as described below, by use of anLY111-immobilized plate and biotinylated LY111.

Each of the assay specimens was added to the wells of the respectiveantibody-immobilized plates described above in an amount of 50 μl/well,followed by incubation at 37° C. for 1 hour. Subsequently, washingsolution was added to the wells in an amount of 200 μl/well, and theplates were washed 4 times.

Avidin-peroxidase (produced by Vector) diluted 1,000-fold with specimendiluting solution (4° C.) and biotinylated LY111 diluted with a specimendiluting solution (4° C.) to attain a concentration of 1.0 μg/ml wereadded to the wells of each antibody-immobilized plate in an amount of 25μl/well each, followed by incubation at 37° C. for 1 hour. Subsequently,washing solution was added to the wells in an amount of 200 μl/well, andthe plates were washed 4 times.

The steps from addition of TMB solution (substrate) up to measurement ofabsorbance were the same as those described in (2) above. The results ofabsorptiometry are shown in FIG. 3.

As shown in FIG. 3, urine samples collected from subjects suffering anyof mucopolysaccharidosis types I to VI were found to exhibit highabsorbance values as compared with urine samples collected from healthysubjects. These results indicate that high CS values exhibited in urinesamples from subjects suffering any of mucopolysaccharidosis types I toVI are attributed to CS. Particularly, high CS values in urine samplesof mucopolysaccharidosis types I, II, III, IV, and VI are surprising,because such high data have never been expected.

Thus, it has now been shown that mucopolysaccharidoses can be detectedby correlating assay results of GAG of a single species (in this case,CS) contained in body fluid (urine) with monopolysaccharidoses.

(5) Detection of Mucopolysaccharidoses Through Measurement of DS

Each specimen was diluted with specimen diluting solution, to therebyprepare diluted specimens (0.5 ml each).

The diluted specimens (which may be called “assay specimens”) weresubjected to a GAG assay as described below, by use of the6-B-6-immobilized plate and biotinylated LY111. “6-B-6” is an antibodycapable of specifically binding to the core protein of proteoglycan(decorin). Accordingly, the target GAG to be measured is DS present inthe proteoglycan (decorin) molecule.

Each of the assay specimens was added to the wells of the respectiveantibody-immobilized plates described above in an amount of 50 μl/well,followed by incubation at 37° C. for 1 hour. Subsequently, washingsolution was added to the wells in an amount of 200 μl/well, and theplates were washed 4 times.

Avidin-peroxidase (produced by Vector) diluted 1,000-fold with specimendiluting solution (4° C.) and biotinylated LY111 diluted with a specimendiluting solution (4° C.) to attain a concentration of 1.0 μg/ml wereadded to the wells of each antibody-immobilized plate in an amount of 25μl/well each, followed by incubation at 37° C. for 1 hour. Subsequently,washing solution was added to the wells in an amount of 200 μl/well, andthe plates were washed 4 times.

The steps from addition of TMB solution (substrate) up to measurement ofabsorbance were the same as those described in (2) above. The results ofabsorptiometry are shown in FIG. 3.

As shown in FIG. 4, urine samples collected from subjects suffering anyof mucopolysaccharidosis types I to VI were found to exhibitsignificantly high absorbance as compared with the urine samplescollected from healthy subjects. These results indicate that high DSvalues exhibited in urine samples from subjects sufferingmucopolysaccharidosis types I to VI are attributed to DS. Particularly,high DS values in urine samples of mucopolysaccharidosis types III andIV are surprising, because such high data have never been expected.

Thus, it has now been shown that mucopolysaccharidoses can be detectedby correlating the assay results of GAG of a single species (in thiscase, DS) contained in body fluid (urine) with mucopolysaccharidoses.

Example 2

An inhibition assay was performed to detect mucopolysaccharidoses inserum or urine samples from dogs or cats.

(1) Specimens, Reagents, Etc. Employed in Example 2 are as Follows.

Specimens:

The specimens employed are serum and urine samples collected frommucopolysaccharidosis type IIIB or VII model animals (dogs);mucopolysaccharidosis type I, VI, or VII model animals (oats); andhealthy animals (dogs and oats not suffering mucopolysaccharidosis).

The standard GAG employed is KS derived from bovine cornea (Sigma Co.).

Antibodies:

The primary antibody employed is anti-KS antibody “5D4” (1/20/5D4; ICNImmunobiologicals). The secondary antibody employed ishorseradish-peroxidase-bound anti-mouse IgG (H+L) (Pierce Co.).

Antigen-Immobilized Plate:

An antigen-immobilized plate (i.e., a plate to which KS is immobilized)was prepared as described below.

0.2 U Chondroitinase ABC (produced by Seikagaku Corporation) was addedto 1 mg KS solution (bovine-cornea-derived KS; Sigma), and the resultantsolution was incubated (2 hours, 37° C.) under shaking, to therebydegrade contaminants such as CS. After treatment with ChondroitinaseABC, the KS solution was diluted and added to the wells of animmunoplate (produced by Nunc) in an amount of 200 μl/well, followed byincubation for 2 hours at roam temperature.

The thus-prepared antigen-immobilized plate can be used immediatelyafter washing, and can also be used even after storage for one month orthereabouts at 4° C.

Reagents, Etc.:

Washing solution: PBS containing 0.05% Tween 20 (pH 5.3)

Specimen Diluting Solution:

-   -   PBS containing 1% BSA and 0.05% Tween 20 (pH 5.3)        (2) Detection of mucopolysaccharidoses through measurement of KS

A specimen (serum or urine) was added to an immunoplate to which noantigens were immobilized, and the specimen was diluted to 140 μl/wellwith specimen diluting solution, followed by addition of a primaryantibody solution (140 μl/well) diluted with specimen diluting solutionso as to attain a concentration of 1/18,000 the original concentration.The thus-prepared plate was incubated overnight at 4° C.

The wells of the antigen-immobilized plate were washed three times withwashing solution, and to the thus-washed wells was added the incubatedspecimen mixture (which had undergone reaction with primary antibodies)in an amount of 200 μl/well, followed by incubation at 4° C. for onehour. Subsequently, the wells were washed three times with washingsolution.

The secondary antibody solution diluted 1,000-fold with specimendiluting solution was added to the specimen mixture in an amount of 200μl/well. The specimen mixture was incubated at room temperature for onehour under shaking. Subsequently, the wells were washed three times withwashing solution.

A TMB solution (substrate, Moss Inc.) was added to the walls of theplate in an amount of 200 μl/well, and under observation of developingcolor of the mixture, the plate was incubated at room temperature.Enzymatic reaction of the mixture was stopped by adding 2M HCl (50μl/well), and the absorbance at 490 nm was measured by use of anabsorptiometer. The concentration of KS was obtained through use of theabsorbance data and the calibration curve which had been prepared inadvance from the absorbance data of the standard sample. The resultsfrom the urine specimens are shown below. The values in parentheses arethose corrected with respect to the concentration of creatinine (Cre).

Urine:

Healthy dogs  121.38 ng/ml  (132.49 ng/mg Cre) MPS type IIIB dogs 380.52 ng/ml  (345.85 ng/mg Cre) MPS type VII dogs 1988.28 ng/ml (435.07 ng/mg Cre) MPS type I cats 3150.90 ng/ml (1057.34 ng/mg Cre)MPS type VI cats 1812.78 ng/ml (1066.34 ng/mg Cre) MPS type VII cats3224.61 ng/ml (1258.63 ng/mg Cre)

The results obtained from the serum specimens are shown below.

Serum:

Healthy dogs 89.1 ng/ml MPS type IIIB dogs 186.2 ng/ml MPS type VII dogs457 ng/ml Healthy cats 120 ng/ml MPS type I cats 396 ng/ml MPS type VIcats 554.4 ng/ml MPS type VII cats 483.3 ng/ml

Thus, it has been confirmed that not only human mucopolysaccharidosiscases, but also urine specimens of animals (non-humans) sufferingmucopolysaccharidosis, show significantly high KS level. Moreover, theKS level has been found to be significantly high not only in urine butalso in blood (serum). Furthermore, it has been confirmed thatmucopolysaccharidoses can be detected not only through the sandwichassay method but also through the inhibition assay method. Particularly,high KS levels in urine and blood (serum) samples frommucopolysaccharidosis type I, III (IIIB), VI, and VII animals aresurprising, as such high data have never been expected.

Thus, the above results also show that mucopolysaccharidoses can bedetected by correlating the assay results of GAG of a single species (inthis case, KS) contained in body fluid (urine or blood) withmucopolysaccharidoses.

Example 3 Mass-Scale Detection of Mucopolysaccharidoses:

An attempt was made to detect mucopolysaccharidoses in a mass scale bythe sandwich method using human urine or plasma as samples. The methodis the same as the “(2) Detection of mucopolysaccharidoses throughmeasurement of KS” in Example 1.

The results of using urine as samples (values corrected for creatinine(Cre) concentration) are shown in FIG. 5, and the results of usingplasma in FIGS. 6 and 7.

In FIG. 5, abbreviations show the following results:

-   Control: result of healthy human (n=67);-   All MPS IVA: result of all samples of mucopolysaccharidosis type IVA    human (regardless of age, n=78);-   Severe: result of mucopolysaccharidosis type IVA (severe type) human    (n=54);-   Milder: result of mucopolysaccharidosis type IVA (mild type) human    (n=11);-   Cont 0-5: result of healthy human (from 0 to less than 5 years;    n=21)-   IVA 0-5: result of mucopolysaccharidosis type IVA human (from 0 to    less than 5 years; n=12),-   Cont 5-10: result of healthy human (from 5 to less than 10 yearn;    n=21);-   IVA 5-10: result of mucopolysaccharidosis type IVA human (from 5 to    less than 10 years; n=28);-   Cont 10-15: result of healthy human (from 10 to less than 15 years;    n=10);-   IVA 10-15: result of mucopolysaccharidosis type IVA human (from 10    to less than 15 years; n=9);-   Cont over 15: result of healthy human (15 years or more; n=29); and-   IVA over 15: result of mucopolysaccharidosis type IVA human (15    years or more; n=18).

In FIG. 6, abbreviations show the following results:

-   All cent: result of healthy human (n=112);-   All MPS: result of all samples of mucopolysaccharidosis human    (regardless of age, n=88);-   I: result of mucopolysaccharidosis type I human (regardless of age,    n=17);-   II: result of mucopolysaccharidosis type II human (regardless of    age, n=11);-   III: result of mucopolysaccharidosis type III human (regardless of    age, n=7);-   IVA: result of mucopolysaccharidosis type IVA human (regardless of    age, n=42);-   IVB: result of mucopolysaccharidosis type IVB human (regardless of    age, n=3):-   VI: result of mucopolysaccharidosis type VI human (regardless of    age, n=2);-   VII: result of mucopolysaccharidosis type VII human (regardless of    age, n=6);-   MLII: result of mucolipidosis type II human (regardless of age,    n=2); and-   MLIII: result of mucolipidosis type III human (regardless of age,    n=3).

In FIG. 7, abbreviations show the following results:

-   All coat: result of healthy human (n=112);-   Cord blood: result of cord blood;-   All MPS: result of all samples of mucopolysaccharidosis human    (regardless of age and type, n=88);-   Cont 1-5: result of healthy human (from 1 to less than 5 years;    n=7);-   MPS 1-5: result of mucopolysaccharidosis human (from 1 to less than    5 years; n=19);-   Cont 5-10: result of healthy human (from 5 to less than 10 years;    n=4);-   MPS 5-10: result of mucopolysaccharidosis human (from 5 to less than    10 years; n=27);-   Cont 10-15: result of healthy human (from 10 to less than 15 years;    n=3);-   MPS 10-15: result of mucopolysaccharidosis human (from 10 to less    than 15 years; n=12);-   Cont over 15: result of healthy human (15 years or more; n=11);-   MPS over 15: result of mucopolysaccharidosis human (15 years or    more; n=14).

Each of the boxes in FIGS. 5 to 7 shows a range of from 25% to 75% ineach group. The bar in each box shows average value. Also, the verticalbars outside the box show a range (a range of from 10% to 90%), and thecircles show those departing this range.

Example 4 Detection of Mucolipidoses:

An attempt was made to detect mucolipidoses by the sandwich method usinghuman serum or urine as samples. The method is the same as the “(2)Detection of mucopolysaccharidoses through measurement of KS” in Example1.

The results of using urine as samples (values corrected for creatinine(Cre) concentration) are shown below. Regarding the “healthy human”, theresults are shown as “average value±SD”.

Urine:

Healthy human 0.208 ± 0.142 ng/mg Cre ML type II human 0.92 ng/mg Cre MLtype II human 0.615 ng/mg Cre ML type III human 1.25 ng/mg Cre ML typeIII human 0.75 ng/mg Cre ML (type unclear) human 0.614 ng/mg Cre

Also, the results of using serum as samples are shown below. Regardingthe “healthy human”, the results are shown as “average value±SD”.

Serum:

Healthy human (cord blood) 44.2 ± 27.87 ng/ml Healthy human (1-3 years)127 ± 23.18 ng/ml Healthy human (4-14 years) 237 ± 58 ng/ml Healthyhuman (18 years or more) 137 ± 51.7 ng/ml ML type II human (0.9 year)263 ng/ml ML type III human (12 years) 1147 ng/ml ML type III human (10years) 743 ng/ml ML type III human (40 years) 340 ng/ml

It was confirmed from the above results that the amount of KS ismarkedly increased in animals of not only mucopolysaccharidoses but alsomucolipidoses.

Accordingly, it was shown by these results that mucolipidoses can bedetected by relating the measured result of a single kind of GAG (KS) ina body fluid (urine or blood) to the mucolipidoses.

Example 5 Detection of GM1 Gangliosidoses, Fucosidoses andGalactosialidoses:

An attempt was made to detect GM1 gangliosidoses, fucosidoses andgalactosialidoses by the sandwich method using human serum or urine assamples. The method is the same as the “(2) Detection ofmucopolysaccharidoses through measurement of KS” in Example 1.

The results of using urine as samples (values corrected for creatinine(Cre) concentration) are shown below. Regarding the “healthy human”, theresults are shown as “average value±SD”.

Urine:

Healthy human 0.215 ± 0.14 ng/mg Cre GM1 gangliosidosis human 3.406ng/mg Cre Fucosidosis human 1.44 ng/mg Cre Fucosidosis human 1.37 ng/mgCre Galactosialidosis human 1.18 ng/mg Cre

It was confirmed from the above results that the amount of KS ismarkedly increased in animals of not only mucopolysaccharidoses andmucolipidoses but also galactosialidoses.

Accordingly, it was shown by these results that GM1 gangliosidoses,fucosidoses or galactosialidoses can be detected by relating themeasured result of a single kind of GAG (KS) in a body fluid (urine orblood) to these diseases.

Example 6 Detection Using HPLC:

Using human urine as samples, detection of GAG was carried out by anHPLC-aided method (disaccharide analysis). The results are shown inTable 2.

Also, the “Total-CS” in Table 2 shows total chondroitin sulfate, the“DS-4S” shows 4-position-sulfated dermatan sulfate and the “Cre” showscreatinine.

TABLE 2 Total-CS Total-CS DS-4S DS-4S KS KS HS HS Cre Disease (μg/mL)μg/mgCre (μg/mL) μg/mgCre (μg/mL) μg/mgCre (μg/mL) μg/mgCre (mg/dL) MPSI 90.7 166.1 246.5 451.3 12.3 22.5 51.6 94.6 55 MPS I 8.6 195.6 16.8383.5 1.1 26.2 5.1 115.7 4 MPS II 40.2 77.1 91.5 175.7 6.8 13.1 46.188.5 52 MPS II 83.1 117.1 143.8 202.6 9.0 12.6 73.0 102.8 71 MPS II 40.4206.4 122.0 623.0 2.7 14.0 27.5 140.2 20 MPS III 7.5 81.3 2.3 25.3 1.313.7 18.6 202.2 9 MPS IIIA 12.5 93.9 2.0 15.0 2.2 16.4 24.5 183.6 13 MPSIIIB 8.3 45.3 1.5 8.1 1.6 8.6 30.0 164.6 18 MPS IIIB 20.1 54.4 2.8 7.52.9 7.8 81.0 219.7 37 MPS IVA 34.1 147.9 2.9 12.4 28.9 125.3 2.2 9.5 23MPS IVA 36.2 167.0 2.1 9.6 25.7 118.7 3.1 14.2 22 MPS IV? 175.2 333.44.2 8.0 56.4 107.4 5.9 11.1 53 MPS IVB 5.5 89.1 1.7 27.7 4.1 65.7 0.35.1 6 MPS IVB 54.4 46.5 3.1 2.7 48.7 41.6 1.0 0.9 117 ML II 22.1 233.41.4 15.0 3.3 34.8 1.1 11.8 9 ML III 64.4 45.2 11.3 7.9 19.4 13.7 11.17.8 142 ML III 11.1 7.9 3.6 2.5 2.8 2.0 2.7 1.9 141 GM I 5.5 59.2 0.22.6 5.4 58.1 0.2 2.5 9 Fucosidosis 50.4 75.2 4.0 6.0 23.7 35.3 3.1 4.667 Fucosidosis 31.6 50.7 1.5 2.4 16.6 26.6 1.7 2.8 62 Healthy person21.1 48.7 0.3 0.6 2.5 5.8 0.4 1.0 43 Healthy parson 9.9 33.2 0.4 1.2 1.85.9 0.3 0.9 30 Healthy person 12.4 22.7 0.5 1.0 1.8 3.4 0.6 1.0 55Healthy parson 30.9 18.2 1.7 1.0 3.2 1.9 3.2 1.9 170 Healthy person 12.57.6 0.4 0.3 0.9 0.6 0.6 0.4 164

It was shown from Table 2 that the amount of GAG is increased in eachdisease. Based on this, it was shown that the method of the inventioncan be carried out also by a method which does not use antibodies.

Example 7 Mass-Scale Detection of Lysosomal Storage Diseases:

An attempt was made to detect lysosomal storage diseases in a mass scaleby the sandwich method using human urine or serum as samples. The methodis the same as the “(3) Detection of mucopolysaccharidoses throughmeasurement of HS” in Example 1.

The results of using serum as samples are shown in below. The resultsare shown as “average value”.

Serum:

Healthy human (n = 51) 4.89 U/ml MPS type I human (n = 16) 38.0 U/ml MPStype II human (n = 25) 82.1 U/ml MPS type IIIA human (n = 6) 22.0 U/mlMPS type IIIB human (n = 6) 26.8 U/ml MPS type IIIC human (n = 3) 13.4U/ml MPS type IVA human (n = 29) 7.51 U/ml MPS type IVB human (n = 2)9.43 U/ml MPS type VI human (n = 3) 12.0 U/ml MPS type VII human (n = 5)18.9 U/ml MLD human (n = 4) 9.82 U/ml LIPO human (n = 1) 103 U/ml TShuman (n = 7) 13.0 U/ml GSD type I human (n = 1) 19.1 U/ml GSD type IIhuman (n = 1) 7.57 U/ml Sandhoff disease human (n = 3) 7.59 U/ml ML typeII human (n = 2) 54.1 U/ml ML type III human (n = 3) 12.2 U/ml NP type Bhuman (n = 5) 8.67 U/ml NP type C human (n = 4) 6.06 U/ml GM2gangliosidoses human (n = 1) 10.7 U/ml Krabbe disease human (n = 3) 6.68U/ml Fabry disease human (n = 5) 10.6 U/ml Gaucher disease type I human(n = 5) 8.18 U/ml Gaucher disease type III human (n = 2) 11.7 U/ml

It was confirmed from the above results that the amount of HS isincreased in animals of not only mucopolysaccharidoses but also severalkinds of lysosomal storage diseases.

Accordingly, it was shown by these results that lysosomal storagediseases can be detected by relating the measured result of a singlekind of GAG (85) in a body fluid (urine or blood) to these diseases.

Example 8 Preparation of a Kit of the Present Invention (1)

A kit of the present invention including the below-described componentswas prepared. The kit can be used to detect mucopolysaccharidoses, etc.through sandwich assay of GAG.

-   -   1. 96-well immunoplate, immobilized with 5D4 . . . 1 plate    -   2. Biotinylated 5D4 1 vial    -   3. Avidin-peroxidase . . . 1 vial    -   4. THE solution . . . 1 vial    -   5. Reaction stopping solution (1N HCl). 1 vial    -   6. Washing solution (PBS containing 0.05% Tween 20)    -   7. Specimen diluting solution (PBS(−) containing 1% bovine serum        albumin (BSA))    -   8. KS standard solutions . . . 1 set

Also, another kit of the present invention was prepared, in which a96-well F58-10E4-immobilized immunoplate and biotinylated F58-10E4 wereprovided instead of the 5D4-immobilized immunoplate and biotinylated5D4, respectively.

Also, still another kit of the present invention was prepared, in whicha 96-well LY111-immobilized immunoplate and biotinylated LY111 wareprovided instead of the 5D4-immobilized immunoplate and biotinylated5D4, respectively.

Also, yet another kit of the present invention was prepared, in which a96-well 6-B-6-immobilised immunoplate and biotinylated LY111 wereprovided instead of the 5D4-immobilized immunoplate and biotinylated5D4, respectively.

Example 9 Preparation of a Kit of the Present Invention (2)

A kit of the present invention including the below-described componentswas prepared. The kit can be used to detect mucopolysaccharidoses, etc.through inhibition assay of GAG.

-   -   1. 96-well immunoplate, immobilized with KS . . . 1 plate    -   2. 5D4 . . . 1 vial    -   3. Peroxidase-bound anti-mouse IgG (H+L) 1 vial    -   4. TMB solution . . . 1 vial    -   5. Reaction stopping solution (1 N HCl). 1 vial    -   6. Washing solution: PBS containing 0.05% Tween 20 (pH 5.3)    -   7. Specimen diluting solution: PBS containing 1% BSA and 0.05%        Tween 20 WS 5.3)    -   8. KS standard solutions . . . 1 set

While the present invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to one ofskill in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof. Allreferences cited herein are incorporated in their entirety.

This application is based on U.S. provisional patent application No.60/376,194 filed on Apr. 30, 2002 and No. 60/441,326 filed on Jan. 22,2003, the entire contents of which are incorporated hereinto byreference.

INDUSTRIAL APPLICABILITY

The method of the present invention provides high utility in practice,because it ensures highly accurate, highly sensitive, convenient, rapid,inexpensive detection of lysosomal storage diseases. In particular,since the method enables detection of lysosomal storage diseases throughmeasurement of GAG of only one species, measurement of GAGs of aplurality of species is no longer necessary, thereby attainingimprovements in convenience, speed, and cost. The kit of the presentinvention is of great use, as it facilitates the method of theinvention, making its performance more convenient and rapid.

If the present method is performed on all newborn infants to detectpotential lysosomal storage diseases in an early newborn stage duringwhich no clinical syndromes of lysosomal storage diseases aremanifested, there can be performed enzyme supplementing treatment,genetic treatment, bone marrow transplantation, or similar treatment, tothereby possibly prevent mental retardation, etc.

The present invention is of great use, since it can be used not only forthe detection of lysosomal storage diseases but also to grasp theclinical conditions, determine therapeutic regimens, confirm the effectsof treatment, observe the pathological course, evaluate pharmaceuticalproduct development, etc.

1-31. (canceled)
 32. A method for determining the risk of a subjecthaving a lysosomal storage disease comprising: detecting the presence ofheparan sulfate in a blood sample from a subject suspected of having alysosomal storage disease, and determining a higher risk of the subjecthaving the lysosomal storage disease when the level of heparan sulfatedetected in the blood sample is elevated compared to a control value ofa healthy subject.
 33. The method of claim 32, wherein said disease is atype I, II, III, IV, VI or VII mucopolysaccharidosis.
 34. The method ofclaim 32, wherein said disease is a type I mucopolysaccharidosis. 35.The method of claim 32, wherein said disease is a type IImucopolysaccharidosis.
 36. The method of claim 32, wherein said diseaseis a type III mucopolysaccharidosis.
 37. The method according to claim32, wherein said disease is a type IV mucopolysaccharidosis.
 38. Themethod according to claim 32, wherein said disease is a type VImucopolysaccharidosis.
 39. The method according to claim 32, whereinsaid disease is a type VII mucopolysaccharidosis.
 40. The methodaccording to claim 32, wherein said lysosomal storage disease ismetachromatic leukodystrophy, a Niemann-Pick disease, Tay-Sachs disease,Sandhoff disease, GM2 gangliosidosis, Krabbe disease, Fabry disease, aGaucher disease, a glycogen storage disease, or lipofuscinosis.
 41. Themethod according to claim 32, wherein the subject is a newborn.
 42. Amethod for determining the risk of a subject having a lysosomal storagedisease other than a mucopolysaccharidosis comprising: detecting thepresence of a glycosaminoglycan of a single species in a urine sample ofa subject, and determining a higher risk of the subject having thelysosomal storage disease when the level of a glycosaminoglycan of asingle species detected in the urine sample is elevated compared to acontrol value of a healthy subject.
 43. The method according to claim42, wherein said glycosaminoglycan of a single species is heparansulfate and said lysosomal storage disease is mucolipidosis II,mucolipidosis III, GM1 gangliosidosis or fucosidosis.
 44. The methodaccording to claim 42, wherein said glycosaminoglycan of a singlespecies is dermatan sulfate and said lysosomal storage disease ismucolipidosis II, mucolipidosis III, GM1 gangliosidosis or fucosidosis.45. The method according to claim 42, wherein said glycosaminoglycan ofa single species is chondroitin sulfate and said lysosomal storagedisease is mucolipidosis II, mucolipidosis III, GM1 gangliosidosis orfucosidosis.
 46. The method according to claim 42, wherein saidglycosaminoglycan of a single species is keratan sulfate and saidlysosomal storage disease is mucolipidosis II, mucolipidosis III, GM1gangliosidosis, fucosidosis, or galactosialidosis.